4. The method of claim 1, wherein the TRAIL polypeptide is in the form of
an oligomer.

5. The method of claim 2, wherein the TRAIL polypeptide is in the form of
an oligomer.

6. The method of claim 3, wherein the TRAIL polypeptide is in the form of
an oligomer.

7. The method of claim 1, wherein the TRAIL polypeptide is administered in
a composition comprising a physiologically acceptable carrier, diluent,
or excipient.

8. The method of claim 2, wherein the TRAIL polypeptide is administered in
a composition comprising a physiologically acceptable carrier, diluent,
or excipient.

9. The method of claim 3, wherein the TRAIL polypeptide is administered in
a composition comprising a physiologically acceptable carrier, diluent,
or excipient.

10. The method of claim 4, wherein the TRAIL polypeptide is administered
in a composition comprising a physiologically acceptable carrier,
diluent, or excipient.

11. The method of claim 5, wherein the TRAIL polypeptide is administered
in a composition comprising a physiologically acceptable carrier,
diluent, or excipient.

12. The method of claim 6, wherein the TRAIL polypeptide is administered
in a composition comprising a physiologically acceptable carrier,
diluent, or excipient.

13. The method of claim 1, wherein said method further includes
administration of a second anti-cancer agent or radiation therapy.

14. The method of claim 2, wherein said method further includes
administration of a second anti-cancer agent or radiation therapy.

15. The method of claim 3, wherein said method further includes
administration of a second anti-cancer agent or radiation therapy.

16. The method of claim 4, wherein said method further includes
administration of a second anti-cancer agent or radiation therapy.

17. The method of claim 5, wherein said method further includes
administration of a second anti-cancer agent or radiation therapy.

18. The method of claim 6, wherein said method further includes
administration of a second anti-cancer agent or radiation therapy.

19. The method of claim 1, wherein the cancer is a leukemia, melanoma or
lymphoma.

20. The method of claim 2, wherein the cancer is a leukemia, melanoma or
lymphoma.

21. The method of claim 3, wherein the cancer is a leukemia, melanoma or
lymphoma.

22. The method of claim 4, wherein the cancer is a leukemia, melanoma or
lymphoma.

23. The method of claim 5, wherein the cancer is a leukemia, melanoma or
lymphoma.

24. The method of claim 6, wherein the cancer is a leukemia, melanoma or
lymphoma.

25. The method of claim 13, wherein the cancer is prostate cancer or colon
cancer.

26. The method of claim 14, wherein the cancer is prostate cancer or colon
cancer.

27. The method of claim 15, wherein the cancer is prostate cancer or colon
cancer.

28. The method of claim 16, wherein the cancer is prostate cancer or colon
cancer.

29. The method of claim 17, wherein the cancer is prostate cancer or colon
cancer.

30. The method of claim 18, wherein the cancer is prostate cancer or colon
cancer.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of Ser. No. 10/900,399 filed Jul.
28, 2004, which is a continuation of application Ser. No. 09/796,581,
filed Feb. 27, 2001, now abandoned, which is a divisional of application
Ser. No. 09/320,424, filed May 26, 1999, now U.S. Pat. No. 6,284,236,
which is a continuation-in-part of application Ser. No. 09/190,046, filed
Nov. 10, 1998, now abandoned, which is a continuation-in-part of
application Ser. No. 09/048,641, filed Mar. 26, 1998, now abandoned,
which is a continuation-in-part of application Ser. No. 08/670,354, filed
Jun. 25, 1996, now U.S. Pat. No. 5,763,223, which is a
continuation-in-part of application Ser. No. 08/548,368, filed Nov. 1,
1995, now abandoned, which is a continuation-in-part of application Ser.
No. 08/496,632, filed Jun. 29, 1995, now abandoned.

BACKGROUND OF THE INVENTION

[0002]The programmed cell death known as apoptosis is distinct from cell
death due to necrosis. Apoptosis occurs in embryogenesis, metamorphosis,
endocrine dependent tissue atrophy, normal tissue turnover, and death of
immune thymocytes (induced through their antigen-receptor complex or by
glucocorticoids) (Itoh et al., Cell 66:233, 1991). During maturation of
T-cells in the thymus, T-cells that recognize self-antigens are destroyed
through the apoptotic process, whereas others are positively selected.
The possibility that some T-cells recognizing certain self epitopes
(e.g., inefficiently processed and presented antigenic determinants of a
given self protein) escape this elimination process and subsequently play
a role in autoimmune diseases has been suggested (Gammon et al.,
Immunology Today 12:193, 1991). A cell surface antigen known as Fas has
been reported to mediate apoptosis and is believed to play a role in
clonal deletion of self-reactive T-cells (Itoh et al., Cell 66:233, 1991;
Watanabe-Fukunage et al., Nature 356:314, 1992). Cross-linking a specific
monoclonal antibody to Fas has been reported to induce various cell lines
to undergo apoptosis (Yonehara et al., J. Exp. Med., 169:1747, 1989;
Trauth et al., Science, 245:301, 1989). However, under certain
conditions, binding of a specific monoclonal antibody to Fas can have a
costimulatory effect on freshly isolated T cells (Alderson et al., J.
Exp. Med. 178:2231, 1993).

[0004]Investigation into the existence and identity of other molecule(s)
that play a role in apoptosis is desirable. Identifying such molecules
would provide an additional means of regulating apoptosis, as well as
providing further insight into the development of self-tolerance by the
immune system and the etiology of autoimmune diseases.

SUMMARY OF THE INVENTION

[0005]The present invention provides a novel cytokine protein, as well as
isolated DNA encoding the cytokine and expression vectors comprising the
isolated DNA. Properties of the novel cytokine, which is a member of the
tumor necrosis factor (TNF) family of ligands, include the ability to
induce apoptosis of certain types of target cells. This protein thus is
designated TNF Related Apoptosis Inducing Ligand (TRAIL). Among the types
of cells that are killed by contact with TRAIL are cancer cells such as
leukemia, lymphoma, and melanoma cells, and cells infected with a virus.

[0006]A method for producing TRAIL polypeptides involves culturing host
cells transformed with a recombinant expression vector that contains
TRAIL-encoding DNA under conditions appropriate for expression of TRAIL,
then recovering the expressed TRAIL polypeptide from the culture.
Antibodies directed against TRAIL polypeptides are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 presents the results of an assay described in example 8. The
assay demonstrated that a soluble human TRAIL polypeptide induced death
of Jurkat cells, which are a leukemia cell line.

[0008]FIG. 2 presents the results of an assay described in example 11.
Contact with a soluble human TRAIL polypeptide induced death of
cytomegalovirus-infected human fibroblasts, whereas non-virally infected
fibroblasts were not killed.

[0009]FIG. 3 depicts a particular fusion protein encoded by an expression
vector of the present invention. The fusion protein comprises (from N- to
C-terminus) a growth hormone-derived leader sequence (SEQ ID NO:19),
followed by a tripeptide encoded by an oligonucleotide employed in vector
construction, a leucine zipper peptide (SEQ ID NO:15), a tripeptide
encoded by an oligonucleotide employed in vector construction, and a
soluble human TRAIL polypeptide (amino acids 95 to 281 of SEQ ID NO:2). A
DNA sequence encoding the fusion protein, and the amino acid sequence of
the fusion protein, are presented in SEQ ID NO:10 and 11, respectively.

[0010]FIG. 4 depicts a fusion protein encoded by another expression vector
of the present invention, comprising (from N- to C-terminus) a
cytomegalovirus-derived leader sequence (amino acids 1 to 29 of SEQ ID
NO:9), followed by a tripeptide encoded by an oligonucleotide employed in
vector construction (amino acids 30 to 32 of SEQ ID NO:9), a leucine
zipper peptide (SEQ ID NO:15), a tripeptide encoded by an oligonucleotide
employed in vector construction, and a soluble human TRAIL polypeptide
(amino acids 95 to 281 of SEQ ID NO:2). A DNA sequence encoding the
fusion protein, and the amino acid sequence of the fusion protein, are
presented in SEQ ID NO:12 and 13, respectively.

[0012]The present invention also provides antibodies that specifically
bind TRAIL proteins. In one embodiment, the antibodies are monoclonal
antibodies.

[0013]The TRAIL protein induces apoptosis of certain types of target
cells, such as transformed cells that include but are not limited to
cancer cells and virally-infected cells. As demonstrated in examples 5,
8, 9, and 10 below, TRAIL induced apoptosis of human leukemia, lymphoma,
and melanoma cell lines. Among the uses of TRAIL is use in killing cancer
cells. TRAIL finds further use in treatment of viral infections.
Infection with cytomegalovirus (CMV) rendered human fibroblasts
susceptible to apoptosis when contacted with TRAIL, whereas uninfected
fibroblasts were not killed through contact with TRAIL (see example 11).

[0015]E. coli strain DH10B cells transformed with a recombinant vector
containing this human TRAIL DNA were deposited with the American Type
Culture Collection on Jun. 14, 1995, and assigned accession no. 69849.
The deposit was made under the terms of the Budapest Treaty. The
recombinant vector in the deposited strain is the expression vector
pDC409 (described in example 5). The vector was digested with SalI and
NotI, and human TRAIL DNA that includes the entire coding region shown in
SEQ ID NO:1 was ligated into the vector.

[0016]DNA encoding a second human TRAIL protein was isolated as described
in example 2. The nucleotide sequence of this DNA is presented in SEQ ID
NO:3, and the amino acid sequence encoded thereby is presented in SEQ ID
NO:4. The encoded protein comprises an N-terminal cytoplasmic domain
(amino acids 1-18), a transmembrane region (amino acids 19-38), and an
extracellular domain (amino acids 39-101).

[0017]The DNA of SEQ ID NO:3 lacks a portion of the DNA of SEQ ID NO:1,
and is thus designated the human TRAIL deletion variant (huTRAILdv)
clone. Nucleotides 18 through 358 of SEQ ID NO:1 are identical to
nucleotides 8 through 348 of the huTRAILdv DNA of SEQ ID NO:3.
Nucleotides 359 through 506 of SEQ ID NO:1 are missing from the cloned
DNA of SEQ ID NO:3. The deletion causes a shift in the reading frame,
which results in an in-frame stop codon after amino acid 101 of SEQ ID
NO:4. The DNA of SEQ ID NO:3 thus encodes a truncated protein. Amino
acids 1 through 90 of SEQ ID NO:2 are identical to amino acids 1 through
90 of SEQ ID NO:4. However, due to the deletion, the C-terminal portion
of the huTRAILdv protein (amino acids 91 through 101 of SEQ ID NO:4)
differs from the residues in the corresponding positions in SEQ ID NO:2.
In contrast to the full length huTRAIL protein, the truncated huTRAILdv
protein does not exhibit the ability to induce apoptosis of the T cell
leukemia cells of the Jurkat cell line.

[0018]DNA encoding a mouse TRAIL protein has also been isolated, as
described in example 3. The nucleotide sequence of this DNA is presented
in SEQ ID NO:5 and the amino acid sequence encoded thereby is presented
in SEQ ID NO:6. The encoded protein comprises an N-terminal cytoplasmic
domain (amino acids 1-17), a transmembrane region (amino acids 18-38),
and an extracellular domain (amino acids 39-291). This mouse TRAIL is 64%
identical to the human TRAIL of SEQ ID NO:2 at the amino acid level. The
coding region of the mouse TRAIL nucleotide sequence is 75% identical to
the coding region of the human nucleotide sequence of SEQ ID NO:1.

[0020]The TRAIL of the present invention is distinct from the protein
known as Fas ligand (Suda et al., Cell, 75:1169, 1993; Takahashi et al.,
International Immunology 6:1567, 1994). Fas ligand induces apoptosis of
certain cell types, via the receptor known as Fas. As demonstrated in
example 5, TRAIL-induced apoptosis of target cells is not mediated
through Fas. The human TRAIL amino acid sequence of SEQ ID NO:2 is about
20% identical to the human Fas ligand amino acid sequence that is
presented in Takahashi et al., supra. The extracellular domain of human
TRAIL is about 28.4% identical to the extracellular domain of human Fas
ligand.

[0021]The amino acid sequences disclosed herein reveal that TRAIL is a
member of the TNF family of ligands (Smith et al. Cell, 73:1349, 1993;
Suda et al., Cell, 75:1169, 1993; Smith et al., Cell, 76:959, 1994). The
percent identities between the human TRAIL extracellular domain amino
acid sequence and the amino acid sequence of the extracellular domain of
other proteins of this family are as follows: 28.4% with Fas ligand,
22.4% with lymphotoxin-β, 22.9% with TNF-α, 23.1% with
TNF-β, 22.1% with CD30 ligand, and 23.4% with CD40 ligand.

[0022]TRAIL was tested for ability to bind receptors of the TNF-R family
of receptors. The binding analysis was conducted using the slide
autoradiography procedure of Gearing et al. (EMBO J. 8:3667, 1989). The
analysis revealed no detectable binding of human TRAIL to human CD30,
CD40, 4-1BB, OX40, TNF-R (p80 form), CD27, or LTβR (also known as
TNFR-RP). The results in example 5 indicate that human TRAIL does not
bind human Fas.

[0023]The TRAIL polypeptides of the present invention include polypeptides
having amino acid sequences that differ from, but are highly homologous
to, those presented in SEQ ID NOS:2 and 6. Examples include, but are not
limited to, homologs derived from other mammalian species, variants (both
naturally occurring variants and those generated by recombinant DNA
technology), and TRAIL fragments that retain a desired biological
activity. Such polypeptides exhibit a biological activity of the TRAIL
proteins of SEQ ID NOS:2 and 6, and preferably comprise an amino acid
sequence that is at least 80% identical (most preferably at least 90%
identical) to the amino acid sequence presented in SEQ ID NO:2 or SEQ ID
NO:6. These embodiments of the present invention are described in more
detail below.

[0024]Conserved sequences located in the C-terminal portion of proteins in
the TNF family are identified in Smith et al. (Cell, 73:1349, 1993, see
page 1353 and FIG. 6); Suda et al. (Cell, 75:1169, 1993, see FIG. 7);
Smith et at. (Cell, 76:959, 1994, see FIG. 3); and Goodwin et al. (Eur.
J. Immunol., 23:2631, 1993, see FIG. 7 and pages 2638-39), hereby
incorporated by reference. Among the amino acids in the human TRAIL
protein that are conserved (in at least a majority of TNF family members)
are those in positions 124-125 (AH), 136 (L), 154 (W), 169 (L), 174 (L),
180 (G), 182 (Y), 187 (Q), 190 (F), 193 (Q), and 275-276 (FG) of SEQ ID
NO:2. Another structural feature of TRAIL is a spacer region between the
C-terminus of the trans-membrane region and the portion of the
extracellular domain that is believed to be most important for biological
activity. This spacer region, located at the N-terminus of the
extracellular domain, consists of amino acids 39 through 94 of SEQ ID
NO:2. Analogous spacers are found in other family members, e.g., CD40
ligand. Amino acids 138 through 153 correspond to a loop between the P
sheets of the folded (three dimensional) human TRAIL protein.

[0025]Provided herein are membrane-bound TRAIL proteins (comprising a
cytoplasmic domain, a transmembrane region, and an extracellular domain)
as well as TRAIL fragments that retain a desired biological property of
the full length TRAIL protein. In one embodiment, TRAIL fragments are
soluble TRAIL polypeptides comprising all or part of the extracellular
domain, but lacking the transmembrane region that would cause retention
of the polypeptide on a cell membrane. Soluble TRAIL proteins are capable
of being secreted from the cells in which they are expressed.
Advantageously, a heterologous signal peptide is fused to the N-terminus
such that the soluble TRAIL is secreted upon expression.

[0026]Soluble TRAIL may be identified (and distinguished from its
non-soluble membrane-bound counterparts) by separating intact cells which
express the desired protein from the culture medium, e.g., by
centrifugation, and assaying the medium (supernatant) for the presence of
the desired protein. The presence of TRAIL in the medium indicates that
the protein was secreted from the cells and thus is a soluble form of the
TRAIL protein. Naturally-occurring soluble forms of TRAIL are encompassed
by the present invention.

[0027]The use of soluble forms of TRAIL is advantageous for certain
applications. Purification of the proteins from recombinant host cells is
facilitated, since the soluble proteins are secreted from the cells.
Further, soluble proteins are generally more suitable for intravenous
administration.

[0028]Examples of soluble TRAIL polypeptides are those containing the
entire extracellular domain (e.g., amino acids 39 to 281 of SEQ ID NO:2
or amino acids 39 to 291 of SEQ ID NO:6). Fragments of the extracellular
domain that retain a desired biological activity are also provided. Such
fragments advantageously include regions of TRAIL that are conserved in
proteins of the TNF family of ligands, as described above.

[0029]Additional examples of soluble TRAIL polypeptides are those lacking
not only the cytoplasmic domain and transmembrane region, but also all or
part of the above-described spacer region. Soluble human TRAIL
polypeptides thus include, but are not limited to, polypeptides
comprising amino acids x to 281, wherein x represents any of the amino
acids in positions 39 through 95 of SEQ ID NO:2. In the embodiment in
which residue 95 is the N-terminal amino acid, the entire spacer region
has been deleted.

[0030]TRAIL fragments, including soluble polypeptides, may be prepared by
any of a number of conventional techniques. A DNA sequence encoding a
desired TRAIL fragment may be subcloned into an expression vector for
production of the TRAIL fragment. The TRAIL-encoding DNA sequence
advantageously is fused to a sequence encoding a suitable leader or
signal peptide. The desired TRAIL-encoding DNA fragment may be chemically
synthesized using known techniques. DNA fragments also may be produced by
restriction endonuclease digestion of a full length cloned DNA sequence,
and isolated by electrophoresis on agarose gels. If necessary,
oligonucleotides that reconstruct the 5' or 3' terminus to a desired
point may be ligated to a DNA fragment generated by restriction enzyme
digestion. Such oligonucleotides may additionally contain a restriction
endonuclease cleavage site upstream of the desired coding sequence, and
position an initiation codon (ATG) at the N-terminus of the coding
sequence.

[0032]As will be understood by the skilled artisan, the transmembrane
region of each TRAIL protein discussed above is identified in accordance
with conventional criteria for identifying that type of hydrophobic
domain. The exact boundaries of a transmembrane region may vary slightly
(most likely by no more than five amino acids on either end) from those
presented above. Computer programs useful for identifying such
hydrophobic regions in proteins are available.

[0033]The TRAIL DNA of the present invention includes cDNA, chemically
synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations
thereof. Genomic TRAIL DNA may be isolated by hybridization to the TRAIL
cDNA disclosed herein using standard techniques. RNA transcribed from the
TRAIL DNA is also encompassed by the present invention.

[0034]A search of the NCBI databank identified five expressed sequence
tags (ESTs) having regions of identity with TRAIL DNA. These ESTs (NCBI
accession numbers T90422, T82085, T10524, R31020, and Z36726) are all
human cDNA fragments. The NCBI records do not disclose any polypeptide
encoded by the ESTs, and do not indicate what the reading frame, if any,
might be. However, even if the knowledge of the reading frame revealed
herein by disclosure of complete TRAIL coding regions is used to express
the ESTs, none of the encoded polypeptides would have the
apoptosis-inducing property of the presently-claimed TRAIL polypeptides.
In other words, if each of the five ESTs were inserted into expression
vectors downstream from an initiator methionine codon, in the reading
frame elucidated herein, none of the resulting expressed polypeptides
would contain a sufficient portion of the extracellular domain of TRAIL
to induce apoptosis of Jurkat cells.

[0036]Due to degeneracy of the genetic code, two DNA sequences may differ,
yet encode the same amino acid sequence. The present invention thus
provides isolated DNA sequences encoding biologically active TRAIL,
selected from DNA comprising the coding region of a native human or
murine TRAIL cDNA, or fragments thereof, and DNA which is degenerate as a
result of the genetic code to the native TRAIL DNA sequence.

[0037]Also provided herein are purified TRAIL polypeptides, both
recombinant and non-recombinant. Variants and derivatives of native TRAIL
proteins that retain a desired biological activity are also within the
scope of the present invention. In one embodiment, the biological
activity of an TRAIL variant is essentially equivalent to the biological
activity of a native TRAIL protein. One desired biological activity of
TRAIL is the ability to induce death of Jurkat cells. Assay procedures
for detecting apoptosis of target cells are well known. DNA laddering is
among the characteristics of cell death via apoptosis, and is recognized
as one of the observable phenomena that distinguish apoptotic cell death
from necrotic cell death. Examples of assay techniques suitable for
detecting death or apoptosis of target cells include those described in
examples 5 and 8 to 11. Another property of TRAIL is the ability to bind
to Jurkat cells.

[0038]TRAIL variants may be obtained by mutations of native TRAIL
nucleotide sequences, for example. A TRAIL variant, as referred to
herein, is a polypeptide substantially homologous to a native TRAIL, but
which has an amino acid sequence different from that of native TRAIL
because of one or a plurality of deletions, insertions or substitutions.
TRAIL-encoding DNA sequences of the present invention encompass sequences
that comprise one or more additions, deletions, or substitutions of
nucleotides when compared to a native TRAIL DNA sequence, but that encode
an TRAIL protein that is essentially biologically equivalent to a native
TRAIL protein.

[0039]The variant amino acid or DNA sequence preferably is at least 80%
identical to a native TRAIL sequence, most preferably at least 90%
identical. The degree of homology (percent identity) between a native and
a mutant sequence may be determined, for example, by comparing the two
sequences using computer programs commonly employed for this purpose. One
suitable program is the GAP computer program, version 6.0, described by
Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the
University of Wisconsin Genetics Computer Group (UWGCG). The GAP program
utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol.
48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,
1981). Briefly, the GAP program defines identity as the number of aligned
symbols (i.e., nucleotides or amino acids) which are identical, divided
by the total number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a unary
comparison matrix (containing a value of 1 for identities and 0 for
non-identities) for nucleotides, and the weighted comparison matrix of
Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by
Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,
National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty
of 3.0 for each gap and an additional 0.10 penalty for each symbol in
each gap; and (3) no penalty for end gaps.

[0040]Alterations of the native amino acid sequence may be accomplished by
any of a number of known techniques. Mutations can be introduced at
particular loci by synthesizing oligonucleotides containing a mutant
sequence, flanked by restriction sites enabling ligation to fragments of
the native sequence. Following ligation, the resulting reconstructed
sequence encodes an analog having the desired amino acid insertion,
substitution, or deletion.

[0041]Alternatively, oligonucleotide-directed site-specific mutagenesis
procedures can be employed to provide an altered gene having particular
codons altered according to the substitution, deletion, or insertion
required. Techniques for making such alterations include those disclosed
by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);
Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat.
Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein.

[0042]Variants may comprise conservatively substituted sequences, meaning
that one or more amino acid residues of a native TRAIL polypeptide are
replaced by different residues, but that the conservatively substituted
TRAIL polypeptide retains a desired biological activity that is
essentially equivalent to that of a native TRAIL polypeptide. Examples of
conservative substitutions include substitution of amino acids that do
not alter the secondary and/or tertiary structure of TRAIL. Other
examples involve substitution of amino acids outside of the
receptor-binding domain, when the desired biological activity is the
ability to bind to a receptor on target cells and induce apoptosis of the
target cells. A given amino acid may be replaced by a residue having
similar physiochemical characteristics, e.g., substituting one aliphatic
residue for another (such as Ile, Val, Leu, or Ala for one another), or
substitution of one polar residue for another (such as between Lys and
Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions,
e.g., substitutions of entire regions having similar hydrophobicity
characteristics, are well known. TRAIL polypeptides comprising
conservative amino acid substitutions may be tested in one of the assays
described herein to confirm that a desired biological activity of a
native TRAIL is retained. DNA sequences encoding TRAIL polypeptides that
contain such conservative amino acid substitutions are encompassed by the
present invention.

[0043]Conserved amino acids located in the C-terminal portion of proteins
in the TNF family, and believed to be important for biological activity,
have been identified. These conserved sequences are discussed in Smith et
al. (Cell, 73:1349, 1993, see page 1353 and FIG. 6); Suda et al. (Cell,
75:1169, 1993, see FIG. 7); Smith et al. (Cell, 76:959, 1994, see FIG.
3); and Goodwin et al. (Eur. J. Immunol., 23:2631, 1993, see FIG. 7 and
pages 2638-39). Advantageously, the conserved amino acids are not altered
when generating conservatively substituted sequences. If altered, amino
acids found at equivalent positions in other members of the TNF family
are substituted.

[0044]TRAIL also may be modified to create TRAIL derivatives by forming
covalent or aggregative conjugates with other chemical moieties, such as
glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent
derivatives of TRAIL may be prepared by linking the chemical moieties to
functional groups on TRAIL amino acid side chains or at the N-terminus or
C-terminus of a TRAIL polypeptide or the extracellular domain thereof.
Other derivatives of TRAIL within the scope of this invention include
covalent or aggregative conjugates of TRAIL or its fragments with other
proteins or polypeptides, such as by synthesis in recombinant culture as
N-terminal or C-terminal fusions. For example, the conjugate may comprise
a signal or leader polypeptide sequence (e.g. the α-factor leader
of Saccharomyces) at the N-terminus of a TRAIL polypeptide. The signal or
leader peptide co-translationally or post-translationally directs
transfer of the conjugate from its site of synthesis to a site inside or
outside of the cell membrane or cell wall.

[0045]TRAIL polypeptide fusions can comprise peptides added to facilitate
purification and identification of TRAIL. Such peptides include, for
example, poly-His or the antigenic identification peptides described in
U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.
One such peptide is the FLAG® peptide,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:7), which is highly
antigenic and provides an epitope reversibly bound by a specific
monoclonal antibody, thus enabling rapid assay and facile purification of
expressed recombinant protein. This sequence is also specifically cleaved
by bovine mucosal enterokinase at the residue immediately following the
Asp-Lys pairing. Fusion proteins capped with this peptide may also be
resistant to intracellular degradation in E. coli.

[0046]A murine hybridoma designated 4E11 produces a monoclonal antibody
that binds the peptide DYKDDDDK (SEQ ID NO:7) in the presence of certain
divalent metal cations (as described in U.S. Pat. No. 5,011,912), and has
been deposited with the American Type Culture Collection under accession
no HB 9259. Expression systems useful for producing recombinant proteins
fused to the FLAG® peptide, as well as monoclonal antibodies that
bind the peptide and are useful in purifying the recombinant proteins,
are available from Eastman Kodak Company, Scientific Imaging Systems, New
Haven, Conn.

[0047]The present invention further includes TRAIL polypeptides with or
without associated native-pattern glycosylation. TRAIL expressed in yeast
or mammalian expression systems may be similar to or significantly
different from a native TRAIL polypeptide in molecular weight and
glycosylation pattern, depending upon the choice of expression system.
Expression of TRAIL polypeptides in bacterial expression systems, such as
E. coli, provides non-glycosylated molecules.

[0048]Glycosylation sites in the TRAIL extracellular domain can be
modified to preclude glycosylation while allowing expression of a
homogeneous, reduced carbohydrate analog using yeast or mammalian
expression systems. N-glycosylation sites in eukaryotic polypeptides are
characterized by an amino acid triplet Asn-X-Y, wherein X is any amino
acid except Pro and Y is Ser or Thr. Appropriate modifications to the
nucleotide sequence encoding this triplet will result in substitutions,
additions or deletions that prevent attachment of carbohydrate residues
at the Asn side chain. Known procedures for inactivating N-glycosylation
sites in proteins include those described in U.S. Pat. No. 5,071,972 and
EP 276,846. A potential N-glycosylation site is found at positions
109-111 in the human protein of SEQ ID NO:2 and at positions 52-54 in the
murine protein of SEQ ID NO:6.

[0049]Alternatively, known procedures such as mutagenesis may be employed
to add glycosylation sites to TRAIL, thereby promoting an increase in the
carbohydrate moieties attached to TRAIL. Such an approach may be taken
when slowing the clearance of TRAIL from the body following in vivo
administration is desired, for example.

[0050]In another example, sequences encoding Cys residues that are not
essential for biological activity can be altered to cause the Cys
residues to be deleted or replaced with other amino acids, preventing
formation of incorrect intramolecular disulfide bridges upon
renaturation. Cysteine residues are found in the human TRAIL protein of
SEQ ID NO:2 at positions 16, 30, 56, 77, and 230; and in the murine TRAIL
protein of SEQ ID NO:6 at positions 22, 60, 81, and 240.

[0051]Among the soluble human TRAIL polypeptides disclosed herein are
fragments of the extracellular domain that lack the spacer region, as
described above. Such spacer-deleted soluble TRAIL polypeptides include
only one cysteine, corresponding to the residue at position 230 of SEQ ID
NO:2. Thus, any disulfide bonds forming from the Cys-230 residue would be
intermolecular, joining two such soluble TRAIL polypeptides. In the
fusion protein of FIG. 3 (SEQ ID NO:11), the TRAIL polypeptide moiety
comprises only one cysteine, at position 202 (which corresponds to the
cysteine residue at position 230 in the full length human TRAIL sequence
of SEQ ID NO:2). In the fusion protein of FIG. 4 (SEQ ID NO:13), the
TRAIL polypeptide comprises only one cysteine, at position 205 (which
corresponds to the Cys-230 residue in SEQ ID NO:2).

[0052]One embodiment of the invention is directed to a TRAIL polypeptide
(or fusion protein comprising a TRAIL polypeptide), in which the cysteine
residue corresponding to the cysteine at position 230 in SEQ ID NO:2 is
deleted or substituted, in order to prevent formation of disulfide bonds
that involve the Cys-230 residue. If substituted, cysteine may be
replaced by any suitable amino acid, whereby a desired biological
activity of TRAIL is maintained. Examples include, but are not limited
to, serine, alanine, glycine, or valine. Altering the number of cysteine
residues to manipulate oligomer formation is discussed further below.

[0053]Other variants are prepared by modification of adjacent dibasic
amino acid residues to enhance expression in yeast systems in which KEX2
protease activity is present. EP 212,914 discloses the use of
site-specific mutagenesis to inactivate KEX2 protease processing sites in
a protein. KEX2 protease processing sites are inactivated by deleting,
adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg
pairs to eliminate the occurrence of these adjacent basic residues.
Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and
conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and
preferred approach to inactivating KEX2 sites. Potential KEX2 protease
processing sites are found at positions 89-90 and 149-150 in the protein
of SEQ ID NO:2, and at positions 85-86, 135-136, and 162-163 in the
protein of SEQ ID NO:6.

[0054]Naturally occurring TRAIL variants are also encompassed by the
present invention. Examples of such variants are proteins that result
from alternative mRNA splicing events (since TRAIL is encoded by a
multi-exon gene) or from proteolytic cleavage of the TRAIL protein,
wherein a desired biological activity is retained. Alternative splicing
of mRNA may yield a truncated but biologically active TRAIL protein, such
as a naturally occurring soluble form of the protein, for example.
Variations attributable to proteolysis include, for example, differences
in the N- or C-termini upon expression in different types of host cells,
due to proteolytic removal of one or more terminal amino acids from the
TRAIL protein. In addition, proteolytic cleavage may release a soluble
form of TRAIL from a membrane-bound form of the protein. Allelic variants
are also encompassed by the present invention.

[0055]Also provided herein are conjugates or fusion proteins comprising a
TRAIL polypeptide and a tumor-targeting moiety. Such embodiments may be
employed in cancer treatment, for example. The TRAIL component may be any
of the various forms of TRAIL disclosed herein, with one example being a
soluble TRAIL polypeptide. Oligomers comprising such fusion proteins also
are contemplated. The conjugates or fusion proteins may additionally
comprise other components of TRAIL-containing fusions, compositions, and
the like that are described herein. Examples of such other components
include, but are not limited to, leucine zipper peptides.

[0056]The tumor-targeting moiety may be any compound that enhances
delivery of TRAIL to a tumor. Such compounds include, but are not limited
to, compounds that selectively bind to cancer cells compared with normal
cells, specifically bind to a particular type of cancer that is to be
treated, or enhance penetration into solid tumors. In one embodiment, the
tumor-targeting moiety is a peptide.

[0057]Examples of tumor-targeting peptides are described in Arap et al.
(Science 279:377, Jan. 16, 1998), and Pasqualini et al. (Nature
Biotechnology 15:542, June 1997), which are hereby incorporated by
reference in their entirety. Arap et al. and Pasqualini et al. report
studies of peptides that "home" to tumors, selectively binding to tumor
vessels and/or to tumor cells. The tripeptides Arg-Gly-Asp, Asn-Gly-Arg
and Gly-Ser-Leu, or peptides comprising such tripeptide sequences, are
contemplated herein for use as tumor targeting moieties as components of
TRAIL fusion proteins.

[0058]Arap et al. and Pasqualini et al., supra, disclose that peptides
comprising the sequence Arg-Gly-Asp (RGD) bind to integrins, including
but not limited to α, integrins. Such integrins have been detected
in tumor vasculature and on a number of tumor cell types.

[0060]One type of fusion protein provided herein comprises a TRAIL
polypeptide and a peptide that binds an integrin associated with a tumor.
Such integrin may be expressed on tumor cells or tumor vessels, for
example. An example of an integrin is an α integrin. Arap et al.,
supra, note that human α integrins are selectively expressed in
human tumor blood vessels.

Oligomers

[0061]The present invention encompasses TRAIL polypeptides in the form of
oligomers, such as dimers, trimers, or higher oligomers. Oligomers may be
formed by disulfide bonds between cysteine residues on different TRAIL
polypeptides, or by non-covalent interactions between TRAIL polypeptide
chains, for example. In other embodiments, oligomers comprise from two to
four TRAIL polypeptides joined via covalent or non-covalent interactions
between peptide moieties fused to the TRAIL polypeptides. Such peptides
may be peptide linkers (spacers), or peptides that have the property of
promoting oligomerization. Leucine zippers and certain polypeptides
derived from antibodies are among the peptides that can promote
oligomerization of TRAIL polypeptides attached thereto, as described in
more detail below. The TRAIL polypeptides preferably are soluble.

[0062]Preparation of Fusion Proteins Comprising Heterologous Polypeptides
Fused to various portions of antibody-derived polypeptides (including the
Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA
88:10535, 1991); Byrn et al. (Nature 344:667, 1990); and Hollenbaugh and
Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current
Protocols in Immunology, Supplement 4, pages 10.19.1-10.19.11, 1992),
hereby incorporated by reference. In one embodiment of the invention, an
TRAIL dimer is created by fusing TRAIL to an Fc region polypeptide
derived from an antibody. The term "Fc polypeptide" includes native and
mutein forms, as well as truncated Fc polypeptides containing the hinge
region that promotes dimerization. The Fc polypeptide preferably is fused
to a soluble TRAIL (e.g., comprising only the extracellular domain).

[0063]A gene fusion encoding the TRAIL/Fc fusion protein is inserted into
an appropriate expression vector. In one embodiment, the Fc polypeptide
is fused to the N-terminus of a soluble TRAIL polypeptide. The TRAIL/Fc
fusion proteins are allowed to assemble much like antibody molecules,
whereupon interchain disulfide bonds form between the Fc polypeptides,
yielding divalent TRAIL. In other embodiments, TRAIL may be substituted
for the variable portion of an antibody heavy or light chain. If fusion
proteins are made with both heavy and light chains of an antibody, it is
possible to form an TRAIL oligomer with as many as four TRAIL
extracellular regions.

[0064]One suitable Fc polypeptide is the native Fc region polypeptide
derived from a human IgG1, which is described in PCT application WO
93/10151, hereby incorporated by reference. Another useful Fc polypeptide
is the Fc mutein described in U.S. Pat. No. 5,457,035. The amino acid
sequence of the mutein is identical to that of the native Fc sequence
presented in WO 93/10151, except that amino acid 19 has been changed from
Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino
acid 22 has been changed from Gly to Ala. This mutein Fc exhibits reduced
affinity for immunoglobulin receptors.

[0065]Alternatively, oligomeric TRAIL may comprise two or more soluble
TRAIL polypeptides joined through peptide linkers. Examples include those
peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated
by reference). Fusion proteins comprising multiple TRAIL polypeptides
separated by peptide linkers may be produced using conventional
recombinant DNA technology.

[0066]Another method for preparing oligomeric TRAIL polypeptides involves
use of a leucine zipper. Leucine zipper domains are peptides that promote
oligomerization of the proteins in which they are found. Leucine zippers
were originally identified in several DNA-binding proteins (Landschulz et
al., Science 240:1759, 1988), and have since been found in a variety of
different proteins. Among the known leucine zippers are naturally
occurring peptides and derivatives thereof that dimerize or trimerize.

[0067]Leucine zippers were originally identified in several DNA-binding
proteins (Landschulz et al., Science 240:1759, 1988). Zipper domain is a
term used to refer to a conserved peptide domain present in these (and
other) proteins, which is responsible for oligomerization of the
proteins. The zipper domain (also referred to herein as an oligomerizing,
or oligomer-forming, domain) comprises a repetitive heptad repeat, often
with four or five leucine residues interspersed with other amino acids.
Examples of zipper domains are those found in the yeast transcription
factor GCN4 and a heat-stable DNA-binding protein found in rat liver
(C/EBP; Landschulz et al., Science 243:1681, 1989). Two nuclear
transforming proteins, fos and jun, also exhibit zipper domains, as does
the gene product of the murine proto-oncogene, c-myc (Landschulz et al.,
Science 240:1759, 1988). The products of the nuclear oncogenes fos and
jun comprise zipper domains preferentially form a heterodimer (O'Shea et
al., Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989).
The zipper domain is necessary for biological activity (DNA binding) in
these proteins.

[0068]The fusogenic proteins of several different viruses, including
paramyxovirus, coronavirus, measles virus and many retroviruses, also
possess zipper domains (Buckland and Wild; Nature 338:547, 1989; Britton,
Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and Human
Retroviruses 6:703, 1990). The zipper domains in these fusogenic viral
proteins are near the transmembrane region of the proteins; it has been
suggested that the zipper domains could contribute to the oligomeric
structure of the fusogenic proteins. Oligomerization of fusogenic viral
proteins is involved in fusion pore formation (Spruce et al, Proc. Natl.
Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been recently
reported to play a role in oligomerization of heat-shock transcription
factors (Rabindran et al., Science 259:230, 1993).

[0069]Zipper domains fold as short, parallel coiled coils. (O'Shea et al.,
Science 254:539; 1991) The general architecture of the parallel coiled
coil has been well characterized, with a "knobs-into-holes" packing as
proposed by Crick in 1953 (Acta Crystallogr. 6:689). The dimer formed by
a zipper domain is stabilized by the heptad repeat, designated
(abcdefg)n according to the notation of McLachlan and Stewart (J.
Mol. Biol. 98:293; 1975), in which residues a and d are generally
hydrophobic residues, with d being a leucine, which line up on the same
face of a helix. Oppositely-charged residues commonly occur at positions
g and e. Thus, in a parallel coiled coil formed from two helical zipper
domains, the "knobs" formed by the hydrophobic side chains of the first
helix are packed into the "holes" formed between the side chains of the
second helix.

[0070]The residues at position d (often leucine) contribute large
hydrophobic stabilization energies, and are important for oligomer
formation (Krystek et al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et
al. recently reported the synthesis of a triple-stranded α-helical
bundle in which the helices run up-up-down (Science 259:1288, 1993).
Their studies confirmed that hydrophobic stabilization energy provides
the main driving force for the formation of coiled coils from helical
monomers. These studies also indicate that electrostatic interactions
contribute to the stoichiometry and geometry of coiled coils. Further
discussion of the structure of leucine zippers is found in Harbury et al.
(Science 262:1401, 26 Nov. 1993).

[0071]Several studies have indicated that conservative amino acids may be
substituted for individual leucine residues with minimal decrease in the
ability to dimerize; multiple changes, however, usually result in loss of
this ability (Landschulz et al., Science 243:1681, 1989; Turner and
Tjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990). van
Heekeren et al. reported that a number of different amino residues can be
substituted for the leucine residues in the zipper domain of GCN4, and
further found that some GCN4 proteins containing two leucine
substitutions were weakly active (Nucl. Acids Res. 20:3721, 1992).
Mutation of the first and second heptadic leucines of the zipper domain
of the measles virus fusion protein (MVF) did not affect syncytium
formation (a measure of virally-induced cell fusion); however, mutation
of all four leucine residues prevented fusion completely (Buckland et
at., J. Gen. Virol. 73:1703, 1992). None of the mutations affected the
ability of MVF to form a tetramer.

[0072]Examples of leucine zipper domains suitable for producing soluble
oligomeric TRAIL proteins include, but are not limited to, those
described in PCT application WO 94/10308 and in U.S. Pat. No. 5,716,805,
hereby incorporated by reference. Recombinant fusion proteins comprising
a soluble TRAIL polypeptide, fused to a peptide that dimerizes or
trimerizes in solution, are expressed in suitable host cells, and the
resulting soluble oligomeric TRAIL is recovered from the culture
supernatant. DNA encoding such fusion proteins is provided herein.

[0076]Other peptides derived from naturally occurring trimeric proteins
may be employed in preparing trimeric TRAIL. Alternatively, synthetic
peptides that promote oligomerization may be employed. In particular
embodiments, leucine residues in a leucine zipper moiety are replaced by
isoleucine residues. Such peptides comprising isoleucine may be referred
to as isoleucine zippers, but are encompassed by the term "leucine
zippers" as employed herein.

[0077]As described in example 7, a soluble Flag®-TRAIL polypeptide
expressed in CV-1/EBNA cells spontaneously formed oligomers believed to
be a mixture of dimers and trimers. The cytotoxic effect of this soluble
Flag®-TRAIL in the assay of example 8 was enhanced by including an
anti-Flag® antibody, possibly because the antibody facilitated
cross-linking of TRAIL/receptor complexes. In one embodiment of the
invention, biological activity of TRAIL is enhanced by employing TRAIL in
conjunction with an antibody that is capable of cross-linking TRAIL.
Cells that are to be killed may be contacted with both a soluble TRAIL
polypeptide and such an antibody.

[0078]As one example, cancer or virally infected cells are contacted with
an anti-Flag® antibody and a soluble Flag®-TRAIL polypeptide.
Preferably, an antibody fragment lacking the Fc region is employed.
Bivalent forms of the antibody may bind the Flag® moieties of two
soluble Flag®-TRAIL polypeptides that are found in separate dimers or
trimers. The antibody may be mixed or incubated with a Flag®-TRAIL
polypeptide prior to administration in vivo. When an LZ-TRAIL protein is
employed, an antibody directed against the leucine zipper peptide may be
substituted for the anti-Flag® antibody, in the foregoing procedures.

[0079]Oligomerization is attributable to factors and mechanisms that
include, but are not limited to, inter-chain disulfide bonds, and
non-covalent interactions such as hydrophobic interactions, as discussed
above. Such factors and mechanisms influence the type of oligomers that
are formed, which may include higher order oligomers, and may result in
protein preparations comprising multiple species of oligomers (e.g.,
dimers, trimers, hexamers, 12-mers, and so on).

[0080]Provided herein are methods for manipulating oligomerization of
TRAIL and TRAIL-containing fusion proteins. The products of these methods
also are provided.

[0081]One approach involves altering the number of cysteine residues in a
TRAIL polypeptide or fusion protein. The number of cysteines may be
increased or decreased, depending upon whether a corresponding increase
or decrease in disulfide bonds is desired.

[0082]One may choose to inhibit or promote disulfide bond formation,
depending on the form of TRAIL that is desired for a particular purpose.
One reason for manipulating disulfide bond formation may be to obtain a
more homogeneous protein preparation, by controlling one mechanism of
oligomerization. The proportion of oligomers that are of a desired
species may be increased through such an approach. Another reason may be
to enhance the proportion of a particularly advantageous form of TRAIL in
a protein preparation, such as an oligomeric form exhibiting enhanced
biological activity.

[0083]The amino acid sequence of a TRAIL protein or fusion protein may be
altered to increase or decrease the number of cysteine residues. Such
sequence alteration may be accomplished by conventional procedures, such
as mutagenesis techniques, as discussed above. One alternative for
increasing the number of cysteine residues involves adding
cysteine-containing peptides, preferably fused to the N-terminus of a
TRAIL polypeptide (or included in a fusion protein comprising TRAIL, such
as an LZ-TRAIL fusion).

[0084]The cysteine residue at position 230 of SEQ ID NO:2 is located
within the extracellular domain, which contains the receptor-binding
region. One embodiment of the invention is directed to TRAIL polypeptides
in which the Cys-230 residue is deleted or substituted. Formation of
disulfide bonds involving the Cys-230 residue, including intramolecular
disulfides which would occur in the extracellular domain containing the
receptor-binding function of the protein, thus is avoided.

[0085]Oligomers may be treated with chemical cross-linking reagents.
Reagents that stabilize the oligomers, without destroying a desired
biological activity, are chosen for use.

Expression Systems

[0086]The present invention provides recombinant expression vectors for
expression of TRAIL, and host cells transformed with the expression
vectors. Any suitable expression system may be employed. The vectors
include a DNA encoding a TRAIL polypeptide, operably linked to suitable
transcriptional or translational regulatory nucleotide sequences, such as
those derived from a mammalian, microbial, viral, or insect gene.
Examples of regulatory sequences include transcriptional promoters,
operators, or enhancers, an mRNA ribosomal binding site, and appropriate
sequences which control transcription and translation initiation and
termination. Nucleotide sequences are operably linked when the regulatory
sequence functionally relates to the TRAIL DNA sequence. Thus, a promoter
nucleotide sequence is operably linked to an TRAIL DNA sequence if the
promoter nucleotide sequence controls the transcription of the TRAIL DNA
sequence. An origin of replication that confers the ability to replicate
in the desired host cells, and a selection gene by which transformants
are identified, are generally incorporated into the expression vector.

[0087]In addition, a sequence encoding an appropriate signal peptide can
be incorporated into expression vectors. A DNA sequence for a signal
peptide (secretory leader) may be fused in frame to the TRAIL sequence so
that the TRAIL is initially translated as a fusion protein comprising the
signal peptide. A signal peptide that is functional in the intended host
cells promotes extracellular secretion of the TRAIL polypeptide. The
signal peptide is cleaved from the TRAIL polypeptide upon secretion of
TRAIL from the cell.

[0088]Suitable host cells for expression of TRAIL polypeptides include
prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts are described, for example, in Pouwels et al. Cloning
Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free
translation systems could also be employed to produce TRAIL polypeptides
using RNAs derived from DNA constructs disclosed herein.

[0089]Prokaryotes include gram negative or gram positive organisms, for
example, E. coli or Bacilli. Suitable prokaryotic host cells for
transformation include, for example, E. coli, Bacillus subtilis,
Salmonella typhimurium, and various other species within the genera
Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host
cell, such as E. coli, a TRAIL polypeptide may include an N-terminal
methionine residue to facilitate expression of the recombinant
polypeptide in the prokaryotic host cell. The N-terminal Met may be
cleaved from the expressed recombinant TRAIL polypeptide.

[0090]Expression vectors for use in prokaryotic host cells generally
comprise one or more phenotypic selectable marker genes. A phenotypic
selectable marker gene is, for example, a gene encoding a protein that
confers antibiotic resistance or that supplies an autotrophic
requirement. Examples of useful expression vectors for prokaryotic host
cells include those derived from commercially available plasmids such as
the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for
ampicillin and tetracycline resistance and thus provides simple means for
identifying transformed cells. An appropriate promoter and a TRAIL DNA
sequence are inserted into the pBR322 vector. Other commercially
available vectors include, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,
USA).

[0092]TRAIL alternatively may he expressed in yeast host cells, preferably
from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of
yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast
vectors will often contain an origin of replication sequence from a 2μ
yeast plasmid, an autonomously replicating sequence (ARS), a promoter
region, sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Suitable promoter sequences
for yeast vectors include, among others, promoters for metallothionein,
3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,
1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.
7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as
enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phospho-glucose isomerase, and glucokinase. Other suitable vectors and
promoters for use in yeast expression are further described in Hitzeman,
EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter
described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et
al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and
E. coli may be constructed by inserting DNA sequences from pBR322 for
selection and replication in E. coli (Ampr gene and origin of
replication) into the above-described yeast vectors.

[0093]The yeast α-factor leader sequence may be employed to direct
secretion of the TRAIL polypeptide. The α-factor leader sequence is
often inserted between the promoter sequence and the structural gene
sequence. See, e.g., Kurjan et at., Cell 30:933, 1982 and Bitter et al.,
Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable
for facilitating secretion of recombinant polypeptides from yeast hosts
are known to those of skill in the art. A leader sequence may be modified
near its 3' end to contain one or more restriction sites. This will
facilitate fusion of the leader sequence to the structural gene.

[0094]Yeast transformation protocols are known to those of skill in the
art. One such protocol is described by Hinnen et al., Proc. Natl. Acad.
Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp.sup.+
transforrnants in a selective medium, wherein the selective medium
consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,
10 μg/ml adenine and 20 μg/ml uracil.

[0095]Yeast host cells transformed by vectors containing an ADH2 promoter
sequence may be grown for inducing expression in a "rich" medium. An
example of a rich medium is one consisting of 1% yeast extract, 2%
peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80
μg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is
exhausted from the medium.

[0097]CHO cells are preferred for use as host cells. One example of a
suitable CHO cell line is the cell line designated DX-B 11, which is
deficient in dihydrofolate reductase (DHFR), as described in Urlaub and
Chasin (Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980), hereby
incorporated by reference. DX-B11 cells may be transformed with
expression vectors that encode DHFR, which serves as a selectable marker
(Kauffman et al., Meth. in Enzymology, 185:487-511, 1990). The use of
DHFR as a selectable marker, when cells are cultured in medium containing
methotrexate, and for amplifying a heterologous DNA inserted into the
expression vector, are well known.

[0098]In other embodiments, the host cells are CHO cells that can be grown
in suspension culture, and that are adapted to grow in media that does
not contain serum. The cells may be further adapted to grow in media
lacking insulin-like growth factor (IGF-1) and/or transferrin. The host
cells may be adapted to grow in media that does not contain any exogenous
growth factors that are animal proteins.

[0099]Such CHO cell lines may be generated by any suitable procedure. One
such procedure is conducted generally as follows. DX-B11 cells are
adapted to growth in serum free medium by a gradual reduction of serum
supplementation in the media, and replacement of serum with low levels of
the growth factors transferrin and insulin-like growth factor (IGF-1), in
an enriched cell growth media. Cells adapted to serum-free medium then
are weaned off transferrin and insulin-like growth factor-1. The
resulting CHO cells maintain vigorous growth and high viability, as well
as a DHFR-deficient phenotype, in serum-free, essentially protein-free,
media.

[0100]Transformed host cells provided herein include, but are not limited
to, host cells in which heterologous DNA, including a TRAIL-encoding
sequence, is inserted into the cell's genomic DNA. Procedures that result
in integration of expression vectors (or portions thereof) into host cell
DNA are well known. Conventional procedures may be employed to amplify,
or increase the copy number of, heterologous DNA integrated into the
genomic DNA of transformed host cells.

[0101]Transcriptional and translational control sequences for mammalian
host cell expression vectors may be excised from viral genomes. Commonly
used promoter sequences and enhancer sequences are derived from Polyoma
virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
DNA sequences derived from the SV40 viral genome, for example, SV40
origin, early and late promoter, enhancer, splice, and polyadenylation
sites may be used to provide other genetic elements for expression of a
structural gene sequence in a mammalian host cell. Viral early and late
promoters are particularly useful because both are easily obtained from a
viral genome as a fragment which may also contain a viral origin of
replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40
fragments may also be used, provided the approximately 250 bp sequence
extending from the Hind III site toward the Bgl I site located in the
SV40 viral origin of replication site is included.

[0102]Expression vectors for use in mammalian host cells can be
constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,
1983), for example. A useful system for stable high level expression of
mammalian cDNAs in C127 murine mammary epithelial cells can be
constructed substantially as described by Cosman et al. (Mol. Immunol.
23:935, 1986). A high expression vector, PMLSV N1/N4, described by Cosman
et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional
mammalian expression vectors are described in EP-A-0367566, and in WO
91/18982. As one alternative, the vector may be derived from a
retrovirus. Additional suitable expression systems are described in the
examples below.

[0103]One preferred expression system employs Chinese hamster ovary (CHO)
cells and an expression vector designated PG5.7. This expression vector
is described in U.S. patent application Ser. No. 08/586,509, filed Jan.
11, 1996, and in PCT application publication no. WO 97/25420, which are
hereby incorporated by reference. PG5.7 components include a fragment of
CHO cell genomic DNA, followed by a CMV-derived promoter, which is
followed by a sequence encoding an adenovirus tripartite leader, which in
turn is followed by a sequence encoding dihydrofolate reductase (DHFR).
These components were inserted into the plasmid vector pGEM1 (Promega,
Madison, Wis.). DNA encoding a TRAIL polypeptide (or fusion protein
containing TRAIL) may be inserted between the sequences encoding the
tripartite leader and DHFR. Methotrexate may be added to the culture
medium to increase expression levels, as is recognized in the field.

[0105]A further example of a suitable expression vector is similar to
PG5.7, but comprises a multiple cloning site and an internal ribosome
binding site (IRES), positioned between the adenovirus tripartite leader
and DHFR-encoding sequences. The multiple cloning site comprises several
restriction endonuclease recognition sites, at which heterologous DNA
(e.g., TRAIL, DNA) may be inserted into the vector. The IRES, a 575 bp
non-coding region derived from the encephalomyocarditis virus, allows
cap-independent internal binding of the ribosome and initiation of
translation. For discussion of the use of IRES sequences in expression
vectors, including the role such sequences play in allowing dicistronic
mRNAs to be translated efficiently, see Kaufman R., Nucleic Acids
Research 19:4485, 1991; Oh and Sarnow, Current Opinion in Genetics and
Development 3:295-300, 1993; and Ramesh et al., Nucleic Acids Research,
24:2697-2700, 1996. In addition, the vector may comprise a truncated CHO
genomic DNA fragment, shorter than the fragment incorporated into PG5.7,
yet still functional in enhancing expression of TRAIL (see WO 97/25420).

[0106]For expression of TRAIL, a type II protein lacking a native signal
sequence, a heterologous signal sequence or leader functional in
mammalian host cells may be added. Examples include the signal sequence
for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, the signal
sequence for interleukin-2 receptor described in Cosman et al., Nature
312:768 (1984); the interleukin-4 receptor signal peptide described in EP
367,566; the type I interleukin-1 receptor signal peptide described in
U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal
peptide described in EP 460,846. Another option is a leader derived from
Ig-kappa (cite), such as a leader comprising the amino acid sequence
Met-Gly-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-Gly-Ser-T-
hr-Gly (SEQ ID NO:25). Further alternatives are cytomegalovirus-derived
leaders and signal peptides derived from a growth hormone, as described
in more detail below.

[0108]In another embodiment of the invention, the FLAG® peptide in the
fusion protein described immediately above is replaced with a leucine
zipper peptide. Thus, one recombinant expression vector provided herein
comprises DNA encoding a fusion protein comprising a CMV leader, a
leucine zipper peptide, and a soluble TRAIL polypeptide. One example of
such a fusion protein is depicted in FIG. 4 (SEQ ID NO:13). The protein
of FIG. 4 comprises (from N- to C-terminus) a CMV leader (residues 1
through 29 of SEQ ID NO:9); an optional tripeptide Thr-Ser-Ser encoded by
oligonucleotides employed in vector construction (residues 30 through 32
of SEQ ID NO:9); a leucine zipper (SEQ ID NO: 15); an optional tripeptide
Thr-Arg-Ser encoded by oligonucleotides employed in vector construction;
and amino acids 95 to 281 of the human TRAIL protein of SEQ ID NO:2.

[0109]Expression systems that employ such CMV-derived leader peptides are
useful for expressing proteins other than TRAIL. Expression vectors
comprising a DNA sequence that encodes amino acids 1 through 29 of SEQ ID
NO:9 are provided herein. In another embodiment, the vector comprises a
sequence that encodes amino acids 1 through 28 of SEQ ID NO:9. DNA
encoding a desired heterologous protein is positioned downstream of, and
in the same reading frame as, DNA encoding the leader. Additional
residues (e.g., those encoded by linkers or primers) may be encoded by
DNA positioned between the sequences encoding the leader and the desired
heterologous protein, as illustrated by the vector described in example
7. As is understood in the pertinent field, the expression vectors
comprise promoters and any other desired regulatory sequences, operably
linked to the sequences encoding the leader and heterologous protein.

[0110]The leader peptide presented in SEQ ID NO:9 may be cleaved after the
arginine residue at position 29 to yield the mature secreted form of a
protein fused thereto. Alternatively or additionally, cleavage may occur
between amino acids 20 and 21, or between amino acids 28 and 29, of SEQ
ID NO:9.

[0111]The skilled artisan will recognize that the position(s) at which the
signal peptide is cleaved may vary according to such factors as the type
of host cells employed, whether murine or human TRAIL is expressed by the
vector, and the like. Analysis by computer program reveals that the
primary cleavage site may be between residues 20 and 21 of SEQ ID NO:9.
Cleavage between residues 22 and 23, and between residues 27 and 28, is
predicted to be possible, as well. To illustrate, expression and
secretion of a soluble murine TRAIL polypeptide resulted in cleavage of a
CMV-derived signal peptide at multiple positions. The three most
prominent species of secreted protein (in descending order) resulted from
cleavage between amino acids 20 and 21 of SEQ ID NO:9, cleavage between
amino acids 22 and 23, and cleavage between amino acids 27 and 28.

[0112]In one particular expression system, in which the fusion protein of
FIG. 4 (SEQ ID NO:13) was expressed in CHO cells, the CMV leader was
cleaved at two positions. Two forms of mature protein resulted, one
comprising amino acids 21 to 256, and the other comprising amino acids 29
to 256, of SEQ ID NO:13.

[0113]A signal peptide comprising amino acids 1 to 20 of the CMV leader of
SEQ ID NO:9 is also provided herein. Such a signal peptide may yield a
more homogeneous preparation of mature protein, since certain of the
above-mentioned alternative signal peptidase cleavage sites are omitted
from the leader.

[0114]A method for producing a heterologous recombinant protein involves
culturing mammalian host cells transformed with such an expression vector
under conditions that promote expression and secretion of the
heterologous protein, and recovering the protein from the culture medium.
Expression systems employing CMV leaders may be used to produce any
desired protein, examples of which include, but are not limited to,
colony stimulating factors, interferons, interleukins, other cytokines,
and cytokine receptors.

[0116]One expression system employing such a signal peptide is described
below, in example 14. In alternative embodiments of the invention, CHO
cells (described above) are employed as host cells, in place of the
CV1-EBNA cells described in example 14. Preferred embodiments of the
present invention are directed to expression vectors encoding a fusion
protein comprising (from N- to C-terminus) a growth hormone leader, the
leucine zipper peptide of SEQ ID NO:15, and a soluble TRAIL polypeptide.
In one embodiment, the TRAIL polypeptide is a soluble human TRAIL
polypeptide comprising amino acids 95 to 281 of SEQ ID NO:2. Optionally,
peptide linkers (which may be encoded by DNA segments resulting from the
vector construction technique, for example) are positioned between the
growth hormone leader and the leucine zipper, or between the leucine
zipper and TRAIL. The leucine zipper moiety promotes oligomerization of
the fusion proteins.

[0117]One example of such a fusion protein is depicted in FIG. 3. The
fusion protein comprises (from N- to C-terminus) a growth hormone-derived
leader sequence (SEQ ID NO:19), followed by a tripeptide encoded by an
oligonucleotide employed in vector construction (Thr-Ser-Ser), a leucine
zipper peptide (SEQ ID NO:15), a tripeptide encoded by an oligonucleotide
employed in vector construction (Thr-Arg-Ser), and a soluble human TRAIL
polypeptide (amino acids 95 to 281 of SEQ ID NO:2). A DNA sequence
encoding the fusion protein, and the amino acid sequence of the fusion
protein, are presented in SEQ ID NO:10 and 11, respectively.

Purified TRAIL Protein

[0118]The present invention provides purified TRAIL proteins, which may be
produced by recombinant expression systems as described above or purified
from naturally occurring cells. The desired degree of purity may depend
on the intended use of the protein. A relatively high degree of purity is
desired when the protein is to be administered in vivo, for example.
Advantageously, TRAIL polypeptides are purified such that no protein
bands corresponding to other proteins are detectable by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized
by one skilled in the pertinent field that multiple bands corresponding
to TRAIL protein may be detected by SDS-PAGE, due to differential
glycosylation, variations in post-translational processing, and the like,
as discussed above. A preparation of TRAIL protein is considered to be
purified as long as no bands corresponding to different (non-TRAIL)
proteins are visualized. TRAIL most preferably is purified to substantial
homogeneity, as indicated by a single protein band upon analysis by
SDS-PAGE. The protein band may be visualized by silver staining,
Coomassie blue staining, or (if the protein is radiolabeled) by
autoradiography.

[0119]One process for producing the TRAIL protein comprises culturing a
host cell transformed with an expression vector comprising a DNA sequence
that encodes TRAIL under conditions such that TRAIL is expressed. The
TRAIL protein is then recovered from the culture (from the culture medium
or cell extracts). As the skilled artisan will recognize, procedures for
purifying the recombinant TRAIL will vary according to such factors as
the type of host cells employed and whether or not the TRAIL is secreted
into the culture medium.

[0120]For example, when expression systems that secrete the recombinant
protein are employed, the culture medium first may be concentrated using
a commercially available protein concentration filter, for example, an
Amicon or Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a purification
matrix such as a gel filtration medium. Alternatively, an anion exchange
resin can be employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose,
dextran, cellulose or other types commonly employed in protein
purification. Alternatively, a cation exchange step can be employed.
Suitable cation exchangers include various insoluble matrices comprising
sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
(e.g., silica gel having pendant methyl or other aliphatic groups) can be
employed to further purify TRAIL. Some or all of the foregoing
purification steps, in various combinations, can be employed to provide a
purified TRAIL protein.

[0121]In one example of a procedure for producing and purifying TRAIL,
Chinese Hamster Ovary (CHO) cells are stably transformed with a
recombinant expression vector encoding soluble TRAIL. In one embodiment,
the vector encodes a fusion protein comprising a CMV-derived leader, a
leucine zipper, and a soluble TRAIL polypeptide, as described in more
detail elsewhere herein. The transformed cells are cultured to allow
expression and secretion of the soluble LZ-TRAIL protein into the culture
supernatant. The culture supernatant then is diluted 5-fold with 20 mM
Tris buffer, pH 8.5, and applied to a Q-Sepharose anion exchange column
(Pharmacia LKB, Uppsala, Sweden) at a ratio of 1 ml supernatant per 0.3
ml bead volume. The flow-through then is passed over a Fractogel®
S-Sepharose cation exchange column (EM Separations, Gibbstown, N.J.) at a
ratio of 1/0.06 ml (v/v), washed with five column volumes of buffer, and
eluted with a salt gradient of 0 to 1.0M NaCl in 20 mM Tris buffer, pH
8.5. Fractions containing the LZ/TRAIL protein are pooled and dialyzed
against Tris Buffered Saline (TBS).

[0122]Another example of a protein purification procedure, slightly
modified from the procedure described immediately above, is as follows.
This procedure may be employed when a leader derived from growth hormone
is substituted for the CMV leader, for example. Chinese Hamster Ovary
(CHO) cells are stably transformed with a recombinant expression vector
encoding GH leader-LZ-TRAIL, and cultured to allow expression and
secretion of the soluble LZ-TRAIL protein into the culture supernatant.
The culture supernatant then is diluted 5-fold with 25 mM Tris buffer, pH
7.0, and applied to a Q-Sepharose anion exchange column (Pharmacia LKB,
Uppsala, Sweden) at a ratio of 1 ml supernatant per 0.3 ml bead volume.
The flow-through was concentrated and buffer exchanged into 10 mM Tris,
pH 7.0, then passed over a Fractogel® S-Sepharose cation exchange
column (EM Separations, Gibbstown, N.J.) at a ratio of 1/0.06 ml (v/v);
washed with five column volumes of buffer; and eluted with a 0 to 0.5M
NaCl gradient in 10 mM Tris buffer, pH 7.0. Fractions containing the
LZ/TRAIL protein were concentrated and applied to an S200 sizing column
(Pharmacia) in 10 mM Tris, pH 7.0, 100rnM NaCl, 10% glycerol.

[0123]When bacterial host cells are employed, the recombinant protein
produced in bacterial culture may be isolated by initial disruption of
the host cells, centrifugation, extraction from cell pellets if an
insoluble polypeptide, or from the supernatant fluid if a soluble
polypeptide, followed by one or more concentration, salting-out, ion
exchange, affinity purification or size exclusion chromatography steps.
Finally, RP-HPLC can be employed for final purification steps. Microbial
cells can be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0125]Alternatively, TRAIL polypeptides can be purified by immunoaffinity
chromatography. An affinity column containing an antibody that binds
TRAIL may be prepared by conventional procedures and employed in
purifying TRAIL. Example 4 describes a procedure for generating
monoclonal antibodies directed against TRAIL.

[0126]Expression of various forms of TRAIL described herein in particular
expression systems may yield protein preparations comprising multiple
species of oligomers (e.g., dimers, trimers, hexamers, 12-mers, and so
on). To illustrate, a mixture of oligomers, including hexamers and
trimers, may result from expression of the fusion protein of SEQ ID NO:11
in COS cells. In another illustrative scenario, involving expression of a
fusion protein comprising a growth hormone leader, an isoleucine zipper
and a spacer-deleted soluble TRAIL polypeptide, 12-mers may be among the
resulting oligomers. If a particular species of oligomer is desired for a
particular use, that species may be isolated using conventional
procedures. An example of a suitable procedure employs size exclusion
chromatography.

[0127]A TRAIL protein (e.g., a fusion protein or oligomer) prepared using
a particular expression system may comprise inter- or infra-molecular
disulfide bonds that are disadvantageous for a particular use of the
protein. In such a case, the TRAIL protein may be treated with a reducing
agent in accordance with conventional techniques. An example of a
suitable procedures comprises treating the protein with 5-10 mM DTT
(dithiothreitol) for 10 minutes at 37° C. Other suitable reducing
agents, such as B-mercaptoethanol (preferably at a concentration of at
least 100 mM in the reaction solution), may be substituted for DTT and
used in accordance with standard procedures.

[0128]To inhibit reoxidation and formation of new disulfide bonds, the
protein may be stored in the presence of a reducing agent. One
alternative involves further treatment of the protein, after the reducing
step, with a sulfhydryl-specific modifying agent. Examples of such agents
are iodoacetamide or iodoacetic acid.

[0129]Treatment with a reducing agent may be conducted when refolding of a
protein into a different conformation is desired. Disulfide bonds,
including intramolecular disulfide bonds, are reduced, and the reducing
agent then removed to allow refolding of the protein. If promoting
disulfide bond formation is desired, oxygen can be bubbled through the
reaction solution.

Properties and Uses of TRAIL

[0130]Programmed cell death (apoptosis) occurs during embryogenesis,
metamorphosis, endocrine-dependent tissue atrophy, normal tissue
turnover, and death of immune thymocytes. Regulation of programmed cell
death is vital for normal functioning of the immune system. To
illustrate, T cells that recognize self-antigens are destroyed through
the apoptotic process during maturation of T-cells in the thymus, whereas
other T cells are positively selected. The possibility that some T-cells
recognizing certain self epitopes (e.g., inefficiently processed and
presented antigenic determinants of a given self protein) escape this
elimination process and subsequently play a role in autoimmune diseases
has been proposed (Gammon et al., Immunology Today 12:193, 1991).

[0131]Insufficient apoptosis has been implicated in certain conditions,
while elevated levels of apoptotic cell death have been associated with
other diseases. The desirability of identifying and using agents that
regulate apoptosis in treating such disorders is recognized (Kromer,
Advances in Immunology, 58:211, 1995; Groux et al., J. Exp. Med. 175:331,
1992; Sachs and Lotem, Blood 82:15, 1993).

[0132]Abnormal resistance of T cells toward undergoing apoptosis has been
linked to lymphocytosis, lymphadenopathy, splenomegaly, accumulation of
self-reactive T cells, autoimmune disease, development of leukemia, and
development of lymphoma (Kromer, supra; see especially pages 214-215).
Conversely, excessive apoptosis of T cells has been suggested to play a
role in lymphopenia, systemic immunodeficiency, and specific
immunodeficiency, with specific examples being virus-induced
immunodeficient states associated with infectious mononucleosis and
cytomegalovirus infection, and tumor-mediated immunosuppression (Kromer,
supra; see especially page 214). Depletion of CD4.sup.+ T cells in
HIV-infected individuals may be attributable to inappropriate
activation-induced cell death (AICD) by apoptosis (Groux et al., J. Exp.
Med. 175:331, 1992).

[0133]As demonstrated in examples 5 and 8, TRAIL induces apoptosis of the
acute T cell leukemia cell line designated Jurkat clone E6-1. TRAIL thus
is a research reagent useful in studies of apoptosis, including the
regulation of programmed cell death. Since Jurkat cells are a leukemia
cell line arising from T cells, the TRAIL of the present invention finds
use in studies of the role TRAIL may play in apoptosis of other
transformed T cells, such as other malignant cell types arising from T
cells.

[0134]TRAIL binds Jurkat cells, as well as inducing apoptosis thereof.
TRAIL did not cause death of freshly isolated murine thymocytes, or
peripheral blood T cells (PBTs) freshly extracted from a healthy human
donor. A number of uses flow from these properties of TRAIL.

[0135]TRAIL polypeptides may be used to purify leukemia cells, or any
other cell type to which TRAIL binds. Leukemia cells may be isolated from
a patient's blood, for example. In one embodiment, the cells are purified
by affinity chromatography, using a chromatography matrix having TRAIL
bound thereto. The TRAIL attached to the chromatography matrix may be a
full length protein, an TRAIL fragment comprising the extracellular
domain, an TRAIL-containing fusion protein, or other suitable TRAIL
polypeptide described herein. In one embodiment, a soluble TRAIL/Fc
fusion protein is bound to a Protein A or Protein G column through
interaction of the Fc moiety with the Protein A or Protein G.
Alternatively, TRAIL may be used in isolating leukemia cells by flow
cytometry.

[0136]The thus-purified leukemia cells are expected to die following
binding of TRAIL, but the dead cells will still bear cell surface
antigens, and may be employed as immunogens in deriving anti-leukemia
antibodies. The leukemia cells, or a desired cell surface antigen
isolated therefrom, find further use in vaccine development.

[0137]Since TRAIL binds and kills leukemia cells (the Jurkat cell line),
TRAIL also may be useful in treating leukemia. A therapeutic method
involves contacting leukemia cells with an effective amount of TRAIL. In
one embodiment, a leukemia patient's blood is contacted ex vivo with an
TRAIL polypeptide. The TRAIL may be immobilized on a suitable matrix.
TRAIL binds the leukemia cells, thus removing them from the patient's
blood before the blood is returned into the patient.

[0138]Alternatively or additionally, bone marrow extracted from a leukemia
patient may be contacted with an amount of TRAIL effective in inducing
death of leukemia cells in the bone marrow. Bone marrow may be aspirated
from the sternum or iliac crests, for example, and contacted with TRAIL
to purge leukemia cells. The thus-treated marrow is returned to the
patient.

[0139]TRAIL also binds to, and induces apoptosis of, lymphoma and melanoma
cells (see examples 5, 9, and 10). Thus, uses of TRAIL that are analogous
to those described above for leukemia cells are applicable to lymphoma
and melanoma cells. TRAIL polypeptides may be employed in treating
cancer, including, but not limited to, leukemia, lymphoma, and melanoma.
In one embodiment, the lymphoma is Burkitt's lymphoma. Table I in example
9 shows that TRAIL had a cytotoxic effect on several Burkitt's lymphoma
cell lines. Epstein-Barr virus is an etiologic agent of Burkitt's
lymphoma.

[0140]TRAIL polypeptides also find use in treating viral infections.
Contact with TRAIL caused death of cells infected with cytomegalovirus,
but not of the same cell type when uninfected, as described in example
11. The ability of TRAIL to kill cells infected with other viruses can be
confirmed using the assay described in example 11. Such viruses include,
but are not limited to, encephalomyocarditis virus, Newcastle disease
virus, vesicular stomatitis virus, herpes simplex virus, adenovirus-2,
bovine viral diarrhea virus, HIV, and Epstein-Barr virus.

[0141]An effective amount of TRAIL is administered to a mammal, including
a human, afflicted with a viral infection. In one embodiment, TRAIL is
employed in conjunction with interferon to treat a viral infection. In
the experiment described in example 11, pretreatment of CMV-infected
cells with γ-interferon enhanced the level of killing of the
infected cells that was mediated by TRAIL. TRAIL may be administered in
conjunction with other agents that exert a cytotoxic effect on cancer
cells or virus-infected cells.

[0142]A wide variety of drugs have been employed in cancer treatment.
Examples include, but are not limited to, cisplatin, taxol, etoposide,
Novantrone® (mitoxantrone), actinomycin D, camptothecin (or water
soluble derivatives thereof), methotrexate, mitomycin (e.g., mitomycin
C), dacarbazine (DTIC), and anti-neoplastic antibiotics such as
doxorubicin and daunomycin. Drugs employed in cancer therapy may have a
cytotoxic or cytostatic effect on cancer cells, or may reduce
proliferation of the malignant cells. Cancer treatment may include
radiation therapy. In particular embodiments, TRAIL may be
co-administered with other proteins in cancer therapy; one such protein
is γ-interferon.

[0143]Among the texts providing guidance for cancer therapy is Cancer,
Principles and Practice of Oncology, 4th Edition, DeVita et al., Eds. J.
B. Lippincott Co., Philadelphia, Pa. (1993). An appropriate therapeutic
approach is chosen according to the particular type of cancer, and other
factors such as the general condition of the patient, as is recognized in
the pertinent field.

[0144]TRAIL may be added to a standard chemotherapy regimen, in treating a
cancer patient. For those combinations in which TRAIL and a second
anti-cancer agent exert a synergistic effect against cancer cells, the
dosage of the second agent may be reduced, compared to the standard
dosage of the second agent when administered alone. A method for
increasing the sensitivity of cancer cells to TRAIL comprises
co-administering TRAIL with an amount of a chemotherapeutic anti-cancer
drug that is effective in enhancing sensitivity of cancer cells to TRAIL.

[0145]Particular embodiments of the invention are directed to
co-administration of TRAIL and methotrexate, etoposide, or mitoxantrone
to a cancer patient, including but not limited to prostate cancer
patients. One such therapeutic method comprises administration of TRAIL
and mitoxantrone (Novantrone®; Immunex Corporation, Seattle, Wash.)
to a prostate cancer patient. For descriptions of mitoxantrone or the use
thereof in treating prostate cancer, see U.S. Pat. Nos. 4,197,249 and
4,278,689; and Moore et al. (J. Clinical Oncology 12:689-694, 1994),
which are hereby incorporated by reference. In an in vitro assay in which
a prostate tumor cell line was contacted with various concentrations of
LZ-TRAIL and Novantrone®, a synergistic effect was seen, in that the
combination of LZ-TRAIL and Novantrone® resulting in enhanced tumor
cell death. A synergistic effect also was seen when TRAIL and
methotrexate were employed in the assay. LZ-TRAIL is a fusion protein
comprising a leucine zipper peptide and a soluble TRAIL polypeptide, as
described in more detail above and in example 14.

[0146]Another embodiment of the invention is directed to contacting
colorectal cancer cells (e.g., colon carcinoma cells) with TRAIL and
camptothecene. Alternatives include contacting colorectal cancer cells
with TRAIL in conjunction with adriamycin (doxorubicin) or mitomycin.

[0147]For in vivo use, derivatives of camptothecene that are more water
soluble would be advantageous. Examples of such water soluble derivatives
are the drugs
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin
(CPT-11; irinotecan) and 9-dimethyl-aminomethyl-10-hydroxycamptothecin
(topotecan). Camptothecene and the two above-described derivatives are
DNA topoisomerase I inhibitors.

[0149]Also provided herein are methods for treating melanoma by
administering TRAIL in conjunction with other therapeutic agent(s). In an
in vitro assay, actinomycin D and cycloheximide were found to enhance the
sensitivity of certain melanoma cell lines to TRAIL. For particular
melanoma cell lines that were resistant to TRAIL-mediated cytotoxicity,
addition of the protein synthesis inhibitors actinomycin D or
cycloheximide rendered the cells more sensitive to TRAIL-induced death.
Thus, one method of the present invention comprises co-administering
TRAIL, together with actinomycin D or cycloheximide, to a melanoma
patient.

[0151]One approach toward increasing sensitivity of cancer cells
(including but not limited to melanoma cells) to TRAIL is inhibiting
expression of FLIP in the target cancer cells. Antisense molecules that
are derived from a FLIP DNA sequence, and that will inhibit FLIP
expression in target cells, may be employed in such an approach.

[0152]As used herein, "co-administration" is not limited to simultaneous
administration. TRAIL may be administered along with other therapeutic
agents, during the course of a treatment regimen. In one embodiment,
administration of TRAIL and other therapeutic agents is sequential. An
appropriate time course may be chosen by the physician, according to such
factors as the nature of a patient's illness, and the patient's
condition.

[0154]In another embodiment, TRAIL is used to kill virally infected cells
in cell preparations, tissues, or organs that are to be transplanted. To
illustrate, bone marrow may be contacted with TRAIL to kill virus
infected cells that may be present therein, before the bone marrow is
transplanted into the recipient.

[0155]The TRAIL of the present invention may be used in developing
treatments for any disorder mediated (directly or indirectly) by
defective or insufficient amounts of TRAIL. A therapeutically effective
amount of purified TRAIL protein is administered to a patient afflicted
with such a disorder. Alternatively, TRAIL DNA sequences may be employed
in developing a gene therapy approach to treating such disorders.
Disclosure herein of native TRAIL nucleotide sequences permits the
detection of defective TRAIL genes, and the replacement thereof with
normal TRAIL-encoding genes. Defective genes may be detected in in vitro
diagnostic assays, and by comparison of the native TRAIL nucleotide
sequence disclosed herein with that of a TRAIL gene derived from a person
suspected of harboring a defect in this gene.

[0156]The present invention provides pharmaceutical compositions
comprising purified TRAIL and a physiologically acceptable carrier,
diluent, or excipient. Suitable carriers, diluents, and excipients are
nontoxic to recipients at the dosages and concentrations employed. Such
compositions may comprise buffers, antioxidants such as ascorbic acid,
low molecular weight (less than about 10 residues) polypeptides,
proteins, amino acids, carbohydrates including glucose, sucrose or
dextrins, chelating agents such as EDTA, glutathione and other
stabilizers and excipients commonly employed in pharmaceutical
compositions. Neutral buffered saline or saline mixed with conspecific
serum albumin are among the appropriate diluents. The composition may be
formulated as a lyophilizate using appropriate excipient solutions (e.g.
sucrose) as diluents.

[0157]For therapeutic use, purified proteins of the present invention are
administered to a patient, preferably a human, for treatment in a manner
appropriate to the indication. Thus, for example, the pharmaceutical
compositions can be administered locally, by intravenous injection,
continuous infusion, sustained release from implants, or other suitable
technique. Appropriate dosages and the frequency of administration will
depend, of course, on such factors as the nature and severity of the
indication being treated, the desired response, the condition of the
patient and so forth.

[0158]The TRAIL protein employed in the pharmaceutical compositions
preferably is purified such that the TRAIL protein is substantially free
of other proteins of natural or endogenous origin, desirably containing
less than about 1% by mass of protein contaminants residual of production
processes. Such compositions, however, can contain other proteins added
as stabilizers, carriers, excipients or co-therapeutics.

[0159]The TRAIL-encoding DNAs disclosed herein find use in the production
of TRAIL polypeptides, as discussed above. Fragments of the TRAIL
nucleotide sequences are also useful. In one embodiment, such fragments
comprise at least about 17 consecutive nucleotides, more preferably at
least 30 consecutive nucleotides, of the human or murine TRAIL DNA
disclosed herein. DNA and RNA complements of said fragments are provided
herein, along with both single-stranded and double-stranded forms of the
TRAIL DNA of SEQ ID NOS:1, 3 and 5.

[0160]Among the uses of such TRAIL nucleic acid fragments are use as a
probe or as primers in a polymerase chain reaction (PCR). As one example,
a probe corresponding to the extracellular domain of TRAIL may be
employed. The probes find use in detecting the presence of TRAIL nucleic
acids in in vitro assays and in such procedures as Northern and Southern
blots. Cell types expressing TRAIL can be identified as well. Such
procedures are well known, and the skilled artisan can choose a probe of
suitable length, depending on the particular intended application. For
PCR, 5' and 3' primers corresponding to the termini of a desired TRAIL
DNA sequence are employed to isolate and amplify that sequence, using
conventional techniques.

[0161]Other useful fragments of the TRAIL nucleic acids are antisense or
sense oligonucleotides comprising a single-stranded nucleic acid sequence
(either RNA or DNA) capable of binding to target TRAIL mRNA (sense) or
TRAIL DNA (antisense) sequences. Such a fragment generally comprises at
least about 14 nucleotides, preferably from about 14 to about 30
nucleotides. The ability to create an antisense or a sense
oligonucleotide, based upon a cDNA sequence for a given protein is
described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and
van der Krol et al., BioTechniques 6:958, 1988.

[0162]Binding of antisense or sense oligonucleotides to target nucleic
acid sequences results in the formation of duplexes that block
translation (RNA) or transcription (DNA) by one of several means,
including enhanced degradation of the duplexes, premature termination of
transcription or translation, or by other means. The antisense
oligonucleotides thus may be used to block expression of TRAIL, proteins.

[0163]Antisense or sense oligonucleotides further comprise
oligonucleotides having modified sugar-phosphodiester backbones (or other
sugar linkages, such as those described in WO91/06629) and wherein such
sugar linkages are resistant to endogenous nucleases. Such
oligonucleotides with resistant sugar linkages are stable in vivo (i.e.,
capable of resisting enzymatic degradation) but retain sequence
specificity to be able to bind to target nucleotide sequences. Other
examples of sense or antisense oligonucleotides include those
oligonucleotides which are covalently linked to organic moieties, such as
those described in WO 90/10448, and other moieties that increases
affinity of the oligonucleotide for a target nucleic acid sequence, such
as poly-(L-lysine). Further still, intercalating agents, such as
ellipticine, and alkylating agents or metal complexes may be attached to
sense or antisense oligonucleotides to modify binding specificities of
the antisense or sense olignucleotide for the target nucleotide sequence.

[0164]Antisense or sense oligonucleotides may be introduced into a cell
containing the target nucleic acid sequence by any gene transfer method,
including, for example, CaPO4-mediated DNA transfection,
electroporation, or other gene transfer vectors such as Epstein-Barr
virus. Antisense or sense oligonucleotides are preferably introduced into
a cell containing the target nucleic acid sequence by insertion of the
antisense or sense oligonucleotide into a suitable retroviral vector,
then contacting the cell with the retrovirus vector containing the
inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors
include, but are not limited to, the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors designated
DCT5A, DCT5B and DCT5C (see PCT Application WO 90/13641). Alternatively,
other promotor sequences may be used to express the oligonucleotide.

[0165]Sense or antisense oligonucleotides may also be introduced into a
cell containing the target nucleotide sequence by formation of a
conjugate with a ligand binding molecule, as described in WO 91/04753.
Suitable ligand binding molecules include, but are not limited to, cell
surface receptors, growth factors, other cytokines, or other ligands that
bind to cell surface receptors. Preferably, conjugation of the ligand
binding molecule does not substantially interfere with the ability of the
ligand binding molecule to bind to its corresponding molecule or
receptor, or block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.

[0166]Alternatively, a sense or an antisense oligonucleotide may be
introduced into a cell containing the target nucleic acid sequence by
formation of an oligonucleotidelipid complex, as described in WO
90/10448. The sense or antisense oligonucleotidelipid complex is
preferably dissociated within the cell by an endogenous lipase.

Antibodies Immunoreactive with TRAIL

[0167]The TRAIL proteins of the present invention, or immunogenic
fragments thereof, may be employed in generating antibodies. The present
invention thus provides antibodies that specifically bind TRAIL, i.e.,
the antibodies bind to TRAIL via the antigen-binding sites of the
antibody (as opposed to non-specific binding).

[0168]Polyclonal and monoclonal antibodies may be prepared by conventional
techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New
York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
Production of monoclonal antibodies that are immunoreactive with TRAIL is
further illustrated in example 4 below.

[0169]Antigen-binding fragments of such antibodies, which may be produced
by conventional techniques, are also encompassed by the present
invention. Examples of such fragments include, but are not limited to,
Fab, F(ab'), and F(ab')2 fragments. Antibody fragments and
derivatives produced by genetic engineering techniques are also provided.

[0170]The monoclonal antibodies of the present invention include chimeric
antibodies, e.g., humanized versions of murine monoclonal antibodies.
Such humanized antibodies may be prepared by known techniques, and offer
the advantage of reduced immunogenicity when the antibodies are
administered to humans. In one embodiment, a humanized monoclonal
antibody comprises the variable region of a murine antibody (or just the
antigen binding site thereof) and a constant region derived from a human
antibody. Alternatively, a humanized antibody fragment may comprise the
antigen binding site of a murine monoclonal antibody and a variable
region fragment (lacking the antigen-binding site) derived from a human
antibody. Procedures for the production of chimeric and further
engineered monoclonal antibodies include those described in Riechmann et
al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et
al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,
May, 1993).

[0171]Among the uses of the antibodies is use in assays to detect the
presence of TRAIL polypeptides, either in vitro or in vivo. The
antibodies find further use in_purifying TRAIL by affinity
chromatography.

[0172]Those antibodies that additionally can block binding of TRAIL to
target cells may be used to inhibit a biological activity of TRAIL. A
therapeutic method involves in vivo administration of such an antibody in
an amount effective in inhibiting a TRAIL-mediated biological activity.
Disorders mediated or exacerbated by TRAIL, directly or indirectly, are
thus treated. Monoclonal antibodies are generally preferred for use in
such therapeutic methods.

[0174]Plasma from patients afflicted with TTP (including HIV and HIV
patients) induces apoptosis of human endothelial cells of dermal
microvascular origin, but not large vessel origin (Laurence et al.,
Blood, 87:3245, Apr. 15, 1996). Plasma of TTP patients thus is thought to
contain one or more factors that directly or indirectly induce apoptosis.
In the assay described in example 13 below, polyclonal antibodies raised
against TRAIL inhibited TTP plasma-induced apoptosis of dermal
microvascular endothelial cells. The data presented in example 13 suggest
that TRAIL is present in the serum of TTP patients, and may play a role
in inducing apoptosis of microvascular endothelial cells.

[0175]Another thrombotic microangiopathy is hemolytic-uremic syndrome
(HUS) (Moake, J. L., Lancet, 343:393, 1994; Melnyk et al., (Arch. Intern.
Med., 155:2077, 1995; Thompson et at., supra). One embodiment of the
invention is directed to use of an anti-TRAIL antibody to treat the
condition that is often referred to as "adult HUS" (even though it can
strike children as well). A disorder known as
childhood/diarrheaassociated HUS differs in etiology from adult HUS.

[0176]Other conditions characterized by clotting of small blood vessels
may be treated using anti-TRAIL antibodies. Such conditions include but
are not limited to the following. Cardiac problems seen in about 5-10% of
pediatric AIDS patients are believed to involve clotting of small blood
vessels. Breakdown of the microvasculature in the heart has been reported
in multiple sclerosis patients. As a further example, treatment of
systemic lupus erythematosus (SLE) is contemplated.

[0177]In one embodiment, a patient's blood or plasma is contacted with an
anti-TRAIL antibody ex vivo. The antibody (preferably a monoclonal
antibody) may be bound to a suitable chromatography matrix by
conventional procedures. The patient's blood or plasma flows through a
chromatography column containing the antibody bound to the matrix, before
being returned to the patient. The immobilized antibody binds TRAIL, thus
removing TRAIL protein from the patient's blood.

[0178]In an alternative embodiment, the antibodies are administered in
vivo, in which case blocking antibodies are desirably employed. Such
antibodies may be identified using any suitable assay procedure, such as
by testing antibodies for the ability to inhibit binding of TRAIL to
target cells. Alternatively, blocking antibodies may be identified in
assays for the ability to inhibit a biological effect of the binding of
TRAIL to target cells. Example 12 illustrates one suitable method of
identifying blocking antibodies, wherein antibodies are assayed for the
ability to inhibit TRAIL-mediated lysis of Jurkat cells.

[0179]The present invention thus provides a method for treating a
thrombotic microangiopathy, involving use of an effective amount of an
antibody directed against TRAIL. Antibodies of the present invention may
be employed in in vivo or ex vivo procedures, to inhibit TRAIL-mediated
damage to (e.g., apoptosis of) microvascular endothelial cells.

[0180]Anti-TRAIL antibodies may be employed in conjunction with other
agents useful in treating a particular disorder. In an in vitro study
reported by Laurence et al. (Blood 87:3245, 1996), some reduction of TTP
plasma-mediated apoptosis of microvascular endothelial cells was achieved
by using an anti-Fas blocking antibody, aurintricarboxylic acid, or
normal plasma depleted of cryoprecipitate.

[0181]Thus, a patient may be treated with an agent that inhibits
Fas-ligand-mediated apoptosis of endothelial cells, in combination with
an agent that inhibits TRAIL-mediated apoptosis of endothelial cells. In
one embodiment, an anti-TRAIL blocking antibody and an anti-FAS blocking
antibody are both administered to a patient afflicted with a disorder
characterized by thrombotic microangiopathy, such as TTP or HUS. Examples
of blocking monoclonal antibodies directed against Fas antigen (CD95) are
described in PCT application publication number WO 95/10540, hereby
incorporated by reference.

[0182]Pharmaceutical compositions comprising an antibody that is
immunoreactive with TRAIL, and a suitable, diluent, excipient, or
carrier, are provided herein. Suitable components of such compositions
are as described above for the compositions containing TRAIL proteins.

[0183]The following examples are provided to illustrate particular
embodiments of the present invention, and are not to be construed as
limiting the scope of the invention.

EXAMPLE 1

Isolation of a Human TRAIL DNA

[0184]DNA encoding a human TRAIL protein of the present invention was
isolated by the following procedure. A TBLASTN search of the dbEST data
base at the National Center for Biological Information (NCBI) was
performed, using the query sequence LVVXXXGLYYVYXQVXF (SEQ ID NO:8). This
sequence is based upon the most conserved region of the TNF ligand family
(Smith et al., Cell, 73:1349, 1993). An expressed sequence tag (EST)
file, GenBank accession number Z36726, was identified using these search
parameters. The GenBank file indicated that this EST was obtained from a
human heart atrium cDNA library.

[0185]Two 30-bp oligonucleotides based upon sequences from the 3' and 5'
ends of this EST file were synthesized. The oligonucleotide from the 3'
end had the sequence TGAAATCGAAAGTATGTTTGGGAATAGATG (complement of
nucleotides 636 to 665 of SEQ ID NO:1) and the 5' oligonucleotide was
TGACGAAGAGAGTATGA ACAGCCCCTGCTG (nucleotides 291 to 320 of SEQ ID NO:1).
The oligonucleotides were 5' end labeled with 32P γ-ATP and
polynucleotide kinase. Two λgt10 cDNA libraries were screened by
conventional methods with an equimolar mixture of these labeled
oligonucleotides as probe. One library was a human heart 5' stretch cDNA
library (Stratagene Cloning Systems, La Jolla, Calif.). The other was a
peripheral blood lymphocyte (PBL) library prepared as follows: PBLs were
obtained from normal human volunteers and treated with 10 ng/ml of OKT3
(an anti-CD3 antibody) and 10 ng/ml of human IL-2 for six days. The PBL
cells were washed and stimulated with 500 ng/ml of ionomycin (Calbiochem)
and 10 ng/ml PMA for 4 hours. Messenger RNA was isolated from the
stimulated PBL cells. cDNA synthesized on the mRNA template was packaged
into λgt10 phage vectors (Gigapak®, Stratagene Cloning Systems,
La Jolla, Calif.).

[0187]From the heart 5' stretch cDNA library, one positive plaque was
obtained out of approximately one million plaques. This clone did not
include the 3' end of the gene. Using the PBL library, approximately 50
positive plaques were obtained out of 500,000 plaques. Fifteen of these
first round positive plaques were picked, and the inserts from the
enriched pools were amplified using oligonucleotide primers designed to
amplify phage inserts. The resulting products were resolved by 1.5%
agarose gel electrophoresis, blotted onto nitrocellulose, and analyzed by
standard Southern blot technique using the 32P-labeled 30-mer
oligonucleotides as probes. The two plaque picks that produced the
largest bands by Southern analysis were purified by secondary screening,
and isolated phage plaques were obtained using the same procedures
described above.

[0188]DNA from the isolated phages was prepared by the plate lysis method,
and the cDNA inserts were excised with EcoRI, purified by electrophoresis
using 1.5% agarose in Tris-Borate-EDTA buffer, and ligated into the
pBluescript® SK(+) plasmid. These inserts were then sequenced by
conventional methods, and the resulting sequences were aligned.

[0190]E. coli strain DH10B cells transformed with a recombinant vector
containing this TRAIL DNA were deposited with the American Type Culture
Collection on Jun. 14, 1995, and assigned accession no. 69849. The
deposit was made under the terms of the Budapest Treaty. The recombinant
vector in the deposited strain is the expression vector pDC409 (described
in example 5). The vector was digested with SalI and NotI, and human
TRAIL DNA that includes the entire coding region shown in SEQ ID NO:1 was
ligated into the digested vector.

EXAMPLE 2

Isolation of DNA Encoding a Truncated TRAIL

[0191]DNA encoding a second human TRAIL protein was isolated as follows.
This truncated TRAIL does not exhibit the ability to induce apoptosis of
Jurkat cells.

[0192]PCR analysis, using the 30-mers described in example 1 as the 5' and
3' primers, indicated that 3 out of 14 of the first round plaque picks in
example 1 contained shorter forms of the TRAIL DNA. One of the shortened
forms of the gene was isolated, ligated into the pBluescript® SK(+)
cloning vector (Stratagene Cloning Systems, La Jolla, Calif.) and
sequenced.

[0194]The DNA of SEQ ID NO:3 lacks nucleotides 359 through 506 of the DNA
of SEQ ID NO:1, and is thus designated the human TRAIL deletion variant
(huTRAILdv) clone. The deletion causes a shift in the reading frame,
which results in an in-frame stop codon after amino acid 101 of SEQ ID
NO:4. The DNA of SEQ ID NO:3 thus encodes a truncated protein. Amino
acids 1 through 90 of SEQ ID NO:2 are identical to amino acids 1 through
90 of SEQ ID NO:4. However, due to the deletion, the C-terminal portion
of the huTRAlLdv protein (amino acids 91 through 101 of SEQ ID NO:4)
differs from the residues in the corresponding positions in SEQ ID NO:2.

[0195]The huTRAILdv protein lacks the above-described conserved regions
found at the C-terminus of members of the TNF family of proteins. The
inability of this huTRAILdv protein to cause apoptotic death of Jurkat
cells further confirms the importance of these conserved regions for
biological activity.

EXAMPLE 3

DNA Encoding a Murine TRAIL

[0196]DNA encoding a murine TRAIL, was isolated by the following
procedure. A cDNA library comprising cDNA derived from the mouse T cell
line 7B9 in the vector KZAP was prepared as described in Mosley et al.
(Cell 59:335, 1989). DNA from the library was transferred onto
nitrocellulose filters by conventional techniques.

[0197]Human TRAIL DNA probes were used to identify hybridizing mouse cDNAs
on the filters. Two separate probes were used, in two rounds of
screening. PCR reaction products about 400 bp in length, isolated and
amplified using the human TRAIL DNA as template, were employed as the
probe in the first round of screening. These PCR products consisted of a
fragment of the human TRAIL coding region. The probe used in the second
round of screening consisted of the entire coding region of the human
TRAIL DNA of SEQ ID NO:1. A random primed DNA labeling kit (Stratagene,
La Jolla, Calif.) was used to radiolabel the probes.

[0198]Hybridization was conducted at 37° C. in 50% formamide,
followed by washing with 1×SSC, 0.1% SDS at 50° C. A mouse
cDNA that was positive in both rounds of screening was isolated.

[0199]The nucleotide sequence of this DNA is presented in SEQ ID NO:5 and
the amino acid sequence encoded thereby is presented in SEQ ID NO:6. The
encoded protein comprises an N-terminal cytoplasmic domain (amino acids
1-17), a transmembrane region (amino acids 18-38), and an extracellular
domain (amino acids 39-291). This mouse TRAIL is 64% identical to the
human TRAIL of SEQ ID NO:2, at the amino acid level. The coding region of
the mouse TRAIL nucleotide sequence is 75% identical to the coding region
of the human nucleotide sequence of SEQ ID NO:1.

[0201]Known techniques for producing monoclonal antibodies include those
described in U.S. Pat. No. 4,411,993. Briefly, mice are immunized with
TRAIL as an immunogen emulsified in complete Freund's adjuvant, and
injected in amounts ranging from 10-100 μg subcutaneously or
intraperitoneally. Ten to twelve days later, the immunized animals are
boosted with additional TRAIL emulsified in incomplete Freund's adjuvant.
Mice are periodically boosted thereafter on a weekly to bi-weekly
immunization schedule. Serum samples are periodically taken by
retro-orbital bleeding or tail-tip excision for testing by dot blot assay
or ELISA (Enzyme-Linked Immuno-sorbent Assay) for TRAIL antibodies.

[0202]Following detection of an appropriate antibody titer, positive
animals are provided one last intravenous injection of TRAIL in saline.
Three to four days later, the animals are sacrificed, spleen cells
harvested, and spleen cells are fused to a murine myeloma cell line such
as NS1 or, preferably, P3x63Ag 8.653 (ATCC CRL 1580). Fusions generate
hybridoma cells, which are plated in multiple microtiter plates in a HAT
(hypoxanthine, aminopterin and thymidine) selective medium to inhibit
proliferation of non-fused cells, myeloma hybrids, and spleen cell
hybrids.

[0203]The hybridoma cells are screened by ELISA for reactivity against
purified TRAIL by adaptations of the techniques disclosed in Engvall et
al. (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Positive
hybridoma cells can be injected intraperitoneally into syngeneic BALB/c
mice to produce ascites containing high concentrations of anti-TRAIL
monoclonal antibodies. Alternatively, hybridoma cells can be grown in
vitro in flasks or roller bottles by various techniques. Monoclonal
antibodies produced in mouse ascites can be purified by ammonium sulfate
precipitation, followed by gel exclusion chromatography. Alternatively,
affinity chromatography based upon binding of antibody to protein A or
protein G can be used, as can affinity chromatography based upon binding
to TRAIL.

EXAMPLE 5

DNA Laddering Apoptosis Assay

[0204]Human TRAIL was expressed and tested for the ability to induce
apoptosis. Oligonucleotides were synthesized that corresponded to the 3'
and 5' ends of the coding region of the human TRAIL gene, with SalI and
NotI restriction sites incorporated at the ends of the oligonucleotides.
The coding region of the human TRAIL gene was amplified by standard PCR
techniques, using these oligonucleotides as primers. The PCR reaction
products were digested with the restriction endonucleases SalI and NotI,
then inserted into Sail/NotI-digested vector pDC409. pDC409 is an
expression vector for use in mammalian cells, but is also replicable in
E. coli cells.

[0205]pDC409 is derived from an expression vector designated pDC406
(described in McMahan et al., EMBO J. 10:2821, 1991, and in PCT
application WO 91/18982, hereby incorporated by reference). pDC406
contains origins of replication derived from SV40, Epstein-Barr virus and
pBR322 and is a derivative of HAV-EO described by Dower et al., J.
Immunol. 142:4314 (1989). pDC406 differs from HAV-EO by the deletion of
an intron present in the adenovirus 2 tripartite leader sequence in
HAV-EO. DNA inserted into a multiple cloning site (containing a number of
restriction endonuclease cleavage sites) is transcribed and translated
using regulatory elements derived from HIV and adenovirus. The vector
also contains a gene that confers ampicillin resistance.

[0206]pDC409 differs from pDC406 in that a Bgl II site outside the mcs has
been deleted so that the mcs Bgl II site is unique. Two Pme 1 sites and
one Srf 1 site have been added to the mcs, and three stop codons (TAG)
have been positioned downstream of the mcs to function in all three
reading frames. A T7 primer/promoter has been added to aid in the DNA
sequencing process.

[0207]The monkey kidney cell line CV-1/EBNA-1 (ATCC CRL 10478) was derived
by transfection of the CV-1 cell line (ATCC CCL 70) with a gene encoding
Epstein-Barr virus nuclear antigen-1 (EBNA-1) that constitutively
expresses EBNA-1 driven from the human CMV intermediate-early
enhancer/promoter, as described by McMahan et al., supra. The EBNA-1 gene
allows for episomal replication of expression vectors, such as pDC409,
that contain the EBV origin of replication.

[0208]CV 1/EBNA cells grown in Falcon T175 flasks were transfected with 15
μg of either "empty" pDC409 or pDC409 containing the human TRAIL
coding region. The transformed cells were cultured for three days at
37° C. and 10% CO2. The cells then were washed with PBS, incubated
for 20 minutes at 37° C. in 50 mM EDTA, scraped off of the flask
with a cells scraper, and washed once in PBS. Next, the cells were fixed
in 1% paraformaldehyde PBS for 10 minutes at 4° C., and washed
3× in PBS.

[0209]Jurkat cells were used as the target cells in this assay, to
determine whether the TRAIL-expressing cells could induce apoptosis
thereof. The Jurkat cell line, clone E6-1, is a human acute T cell
leukemia cell line available from the American Type Culture Collection
under accession no. ATCC TIB 152, and described in Weiss et al. (J.
Immunol. 133:123-128, 1984). The Jurkat cells were cultured in RPMI media
supplemented with 10% fetal bovine serum and 10 μg/ml streptomycin and
penicillin to a density of 200,000 to 500,000 cells per ml. Four million
of these cells per well were co-cultured in a 6 well plate with 2.5 mls
of media with various combinations of fixed cells, supernatants from
cells transfected with Fas ligand, and various antibodies, as indicated
below.

[0210]After four hours the cells were washed once in PBS and pelleted at
1200 RPM for 5 minutes in a desktop centrifuge. The pellets were
resuspended and incubated for ten minutes at 4° C. in 500 μl of
buffer consisting of 10 mM Tris-HCl, 10 mM EDTA, pH 7.5, and 0.2% Triton
X-100, which lyses the cells but leaves the nuclei intact. The lysate was
then spun at 4° C. for ten minutes in a micro-centrifuge at 14,000
RPM. The supernatants were removed and extracted three times with 1 ml of
25:24:1 phenol-chloroform-isoamyl alcohol, followed by precipitation with
NaOAC and ethanol in the presence of 1 μg of glycogen carrier (Sigma).

[0212]The results were as follows. Fixed CV1/EBNA cells transfected with
either pDC409 or pDC409-TRAIL produced no detectable DNA laddering.
pDC409-TRAIL fixed cells co-cultured with Jurkat cells produced DNA
laddering, but pDC409 fixed cells co-cultured with Jurkat cells did not.

[0213]DNA laddering was also seen when Jurkat cells were co-cultured with
concentrated supernatants from COS cells transfected with DNA encoding
human Fas ligand in pDC409. The supernatants are believed to contain
soluble Fas ligand that is proteolytically released from the cell
surface. The Fas ligand-induced DNA laddering could be blocked by adding
10 μg/ml of a soluble blocking monoclonal antibody directed against
Fas. This same antibody could not inhibit laddering of Jurkat DNA by the
pDC409-TRAIL cells, which indicates that TRAIL does not induce apoptosis
through Fas.

[0214]In the same assay procedure, fixed CV 1/EBNA cells transfected with
pDC409-TRAIL induced DNA laddering in U937 cells. U937 (ATCC CRL 1593) is
a human histiocytic lymphoma cell line. The ratio of effector to target
cells was 1:4 (the same as in the assay on Jurkat target cells).

[0215]The fragmentation of cellular DNA into a pattern known as DNA
laddering is a hallmark of apoptosis. In the foregoing assay, TRAIL
induced apoptosis of a leukemia cell line and a lymphoma cell line.

EXAMPLE 6

Northern Blot Analysis

[0216]Expression of TRAIL in a number of different tissue types was
analysed in a conventional northern blot procedure. Northern blots
containing poly A.sup.+ RNA from a variety of adult human tissues
(multiple tissue northern blots I and H) were obtained from Clonetech
(Palo Alto, Calif.). Other blots were prepared by resolving RNA samples
on a 1.1% agarose-formaldehyde gel, blotting onto Hybond-N as recommended
by the manufacturer (Amersham Corporation), and staining with methylene
blue to monitor RNA concentrations. The blots were probed with an
antisense RNA riboprobe corresponding to the entire coding region of
human TRAIL.

[0218]TRAIL mRNA was not detected in testis, brain, or liver, or in
several T cell lines. Little or no TRAIL transcripts were detected in
freshly isolated peripheral blood T cells (PBT), either unstimulated or
stimulated with PMA and calcium ionophore for 20 hours.

[0221]The TRAIL-encoding DNA fragment was isolated and amplified by
polymerase chain reaction (PCR), using oligonucleotide primers that
defined the termini of a DNA fragment encoding amino acids 95-281 of SEQ
ID NO:2. The 3' primer was a 31-mer that additionally added a NotI site
downstream of the TRAIL encoding sequence. The 5' primer added an SpeI
site and a Flag® epitope encoding sequence upstream of the
TRAIL-encoding sequence. PCR was conducted by conventional procedures,
using the above-described human TRAIL cDNA as the template.

[0222]The reaction products were digested with SpeI and NotI, and inserted
into the expression vector pDC409 (described in example 5), which had
been cleaved with SalI and NotI. Annealed oligonucleotides that form a
SalI-SpeI fragment encoding a CMV open reading frame leader were also
ligated into the vector. The amino acid sequence of the CMV-derived
leader is presented as SEQ ID NO:9. Amino acids 1 to 29 of SEQ ID NO:9
are encoded by CMV DNA, whereas amino acids 30 to 32 are encoded by
oligonucleotides employed in constructing the vector. E. coli cells were
transfected with the ligation mixture, and the desired recombinant
expression vector was isolated therefrom.

[0223]CV1-EBNA cells (ATCC CRL 10478; described in example 5) were
transfected with the recombinant vector, which is designated
pDC409-Flag-shTRAIL, and cultured to allow expression and secretion of
the soluble Flag®-TRAIL polypeptide. Culture supernatants were
harvested 3 days after transfection and applied to a column containing an
anti-Flag® antibody designated M2 immobilized on a solid support. The
column then was washed with PBS. The monoclonal antibody M2 is described
in Hopp et at., supra, and available from Kodak Scientific Imaging
Systems, New Haven, Conn. 800 μl fractions were eluted from the column
with 50 mM citrate, and immediately neutralized in 0.45 ml 1M Tris (pH
8). Fractions were adjusted to 10% glycerol and stored at -20° C.
until needed.

[0224]This soluble recombinant Flag®/human TRAIL expressed in CV
1/EBNA cells has an apparent molecular weight of 28 kD when analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The Flag® moiety
contributes an estimated 880 daltons to the total molecular weight. Gel
filtration analysis of purified soluble Flag®V/TRAIL suggests that
the molecule is multimeric in solution with a size of -80 kD. While not
wishing to be bound by theory, the gel filtration analysis suggests that
the soluble recombinant Flag®/human TRAIL naturally formed a
combination of dimers and trimers, with trimers predominating.

[0225]An expression vector designated pDC409-Flag-smTRAIL, which encodes a
CMV leader-Flag®-soluble murine TRAIL protein, was constructed by
analogous procedures. A DNA fragment encoding a soluble murine TRAIL
polypeptide was isolated and amplified by PCR. Oligonucleotides that
defined the termini of DNA encoding amino acids 99 to 291 of the murine
TRAIL sequence of SEQ ID NO:6 were employed as the 5' and 3' primers in
the PCR.

[0227]Jurkat cells were cultured to a density of 200,000 to 500,000 cells
per ml in RPMI medium supplemented with 10% fetal bovine serum, 100
μg/ml streptomycin, and 100 μg/ml penicillin. The cells (in 96-well
plates at 50,000 cells per well in a volume of 100 μl) were incubated
for twenty hours with the reagents indicated in FIG. 1. "TRAIL supe."
refers to conditioned superuatant (10 μl per well) from CV1/EBNA cells
transfected with pDC409-Flag-shTRAIL (see example 7). "Control supe."
refers to supernatant from CV1/EBNA cells transfected with empty vector.
Where indicated, immobilized anti-Flag® antibody M2 ("1 mm. M2") was
added at a concentration of 10 μg/ml in a volume of 100 μl per well
and allowed to adhere either overnight at 4° C. or for 2 hours at
37° C., after which wells were aspirated and washed twice with PBS
to remove unbound antibody. Jurkat cells treated with Fas ligand or M3, a
blocking monoclonal antibody directed against Fas, (Alderson et al., J.
Exp. Med. 181:71, 1995; and PCT application WO 95/10540) were included in
the assay as indicated.

[0228]Metabolic activity of the thus-treated cells was assayed by
metabolic conversion of alamar Blue dye, in the following procedure.
Alamar Blue conversion was measured by adding 10 μl of alamar Blue dye
(Biosource International, Camarillo, Calif.) per well, and subtracting
the optical density (OD) at 550-600 nm at the time the dye was added from
the OD 550-600 nm after four hours. No conversion of dye is plotted as 0
percent viability, and the level of dye conversion in the absence of
TRAIL is plotted as 100 percent viability. Percent viability was
calculated by multiplying the ratio of staining of experimental versus
control cultures by 100.

[0229]The results are presented in FIG. 1. Error bars represent the
standard deviation of measurements from four independent wells, and the
values are the average of these measurements.

[0230]The TRAIL-containing supernatant caused a significant reduction in
viability of Jurkat cells. A greater reduction of cell viability resulted
from contact with a combination of TRAIL-containing supernatant and
immobilized anti-Flag® antibody M2. One possible explanation is that
M2 facilitates cross-linking of the Flag®/TRAIL-receptor complexes,
thereby increasing the strength of signaling.

[0231]Fas ligand demonstrated the ability to kill Jurkat cells. The
anti-Fas antibody M3 inhibited the activity of Fas ligand, but not the
activity of TRAIL.

[0232]In order to confirm that the changes in dye conversion in the alamar
Blue assay were due to cell death, the decrease in cell viability induced
by TRAIL was confirmed by staining the cells with trypan blue.

EXAMPLE 9

Lysis of Leukemia and Lymphoma Cells

[0233]In examples 5 and 8, TRAIL induced apoptosis of a leukemia cell line
(Jurkat) and a lymphoma cell line (U937). The following study further
demonstrates the ability of TRAIL to kill leukemia and lymphoma cells.

[0234]The human cell lines indicated in Table I were cultured to a density
of 200,000 to 500,000 cells per ml in RPMI medium supplemented with 10%
fetal bovine serum, 100 μg/ml streptomycin, and 100 μg/ml
penicillin. The cells (in 96-well plates at 50,000 cells per well in a
volume of 100 μl) were incubated for twenty hours with conditioned
supernatants (10 μl per well) from pDC409-Flag-shTRAIL transfected
CVI/EBNA cells.

[0235]Metabolic activity was assayed by conversion of alamar Blue dye, in
the assay procedure described in example 8. The results are presented in
Table I.

[0236]In order to confirm that the changes in dye conversion in the alamar
Blue assay were due to cell death, the decrease in cell viability induced
by TRAIL was confirmed by staining the cells with trypan blue. Crystal
violet staining, performed as described by Flick and Gifford (J. Immunol.
Methods 68:167-175, 1984), also confirmed the results seen in the alamar
Blue assay. The apoptotic nature of the cell death was confirmed by
trypan blue staining and visualization of apoptotic fragmentation by
microscopy.

[0237]As shown in Table I, many cancer cell lines were sensitive to TRAIL
mediated killing. The susceptibility of additional cell types to TRAIL
mediated apoptosis can be determined using the assay procedures described
in this examples section.

[0238]TRAIL exhibited no significant cytotoxic effect on the cell lines
THP-1, K562, Karpas 299, and MP-1. K299, also known as Karpas 299,
(DSM-ACC31) was established from peripheral blood of a male diagnosed
with high grade large cell anaplastic lymphoma (Fischer et al., Blood,
72:234, 1988). MP-1 is a spontaneously derived EBV-transformed B cell
line (Goodwin et al., Cell 73:447, 1993). While not wishing to be bound
by theory, it is possible that these four cell lines do not express a
receptor for TRAIL, or are characterized by upregulation of a gene that
inhibits apoptosis.

[0239]Interspecies cross-reactivity of human and murine TRAIL was tested
as follows. Murine and human TRAIL were incubated with the human melanoma
cell line A375 (ATCC CRL 1619). Since this is an adherent cell line, a
crystal violet assay, rather than alamar Blue, was used to determine cell
viability. A375 cells were cultured in DMEM supplemented with 10% fetal
bovine serum, 100 μg/ml streptomycin, and 100 μg/ml penicillin. The
cells (in 96-well plates at 10,000 cells per well in a volume of 100
μl) were incubated for 72 hours with the soluble murine TRAIL
described in example 7. Crystal violet staining was performed as
described by (Flick and Gifford (J. Immunol. Methods 68:167-175, 1984).
The results demonstrated that both human and murine TRAIL are active on
these human cells, in that murine and human TRAIL killed A375 cells.

[0240]The ability of human TRAIL to act on murine cells was tested, using
the immortalized murine fibroblast cell line L929. Incubation of L929
cells with either human or murine TRAIL resulted in a decrease in crystal
violet staining, thus demonstrating that human and murine TRAIL are
active on (induced apoptosis of) murine cells. In addition to crystal
violet, cell death was confirmed by trypan-blue staining.

EXAMPLE 11

Lysis of CMV-Infected Cells

[0241]The following experiment demonstrates that the soluble
Flag®-human TRAIL protein prepared in example 7 has a cytotoxic
effect on virally infected cells.

[0242]Normal human gingival fibroblasts were grown to confluency on 24
well plates in 10% CO2 and DMEM medium supplemented with 10% fetal
bovine serum, 100 μg/ml streptomycin, and 100 pg/ml penicillin.
Samples of the fibroblasts were treated as indicated in FIG. 2.
Concentrations of cytokines were 10 ng/ml for y-interferon and 30 ng/ml
of soluble Flag®-human TRAIL. All samples receiving TRAIL also
received a two-fold excess by weight of anti-Flag® antibody M2
(described above), which enhances TRAIL activity (presumably by
crosslinking).

[0243]Pretreatment of cells with the indicated cytokines was for 20 hours.
To infect cells with cytomegalovirus (CMV), culture media were aspirated
and the cells were infected with CMV in DMEM with an approximate MOI
(multiplicity of infection) of 5. After two hours the virus containing
media was replaced with DMEM and cytokines added as indicated. After 24
hours the cells were stained with crystal violet dye as described (Flick
and Gifford, 1984, supra). Stained cells were washed twice with water,
disrupted in 200 μl of 2% sodium deoxycholate, diluted 5 fold in
water, and the OD taken at 570 nm. Percent maximal staining was
calculated by normalizing ODs to the sample that showed the greatest
staining. Similar results were obtained from several independent
experiments.

[0244]The results presented in FIG. 2 demonstrate that TRAIL specifically
killed CMV infected fibroblasts. This cell death was enhanced by
pretreatment of the cells with γ-interferon. No significant death
of non-virally infected fibroblasts resulted from contact with TRAIL.

EXAMPLE 12

Assay to Identify Blocking Antibodies

[0245]Blocking antibodies directed against TRAIL may be identified by
testing antibodies for the ability to inhibit a particular biological
activity of TRAIL. In the following assay, a monoclonal antibody was
tested for the ability to inhibit TRAIL-mediated apoptosis of Jurkat
cells. The Jurkat cell line is described in example 5.

[0246]A hybridoma cell line producing a monoclonal antibody raised against
a Flag®/soluble human TRAIL fusion protein was employed in the assay.
Supernatants from the hybridoma cultures were incubated with 20 ng/ml
Flag®/soluble human TRAIL crosslinked with 40 ng/ml anti-Flag®
monoclonal antibody M2, in RPMI complete media in a 96 well microtiter
plate. An equivalent amount of fresh hybridoma culture medium was added
to control cultures. The Flag®/soluble human TRAIL fusion protein and
the monoclonal antibody designated M2 are described in example 7.

[0247]The hybridoma supernatant was employed at a 1:50 (v/v) dilution
(starting concentration), and at two fold serial dilutions thereof. After
incubation at 37° C., 10% CO2, for 30 minutes, 50,000 Jurkat
cells were added per well, and incubation 25 was continued for 20 hours.

[0248]Cell viability was then assessed measuring metabolic conversion of
alamar blue dye. An alamar blue conversion assay procedure is described
in example 8. The monoclonal antibody was found to inhibit the apoptosis
of Jurkat cells induced by Flag®/soluble human TRAIL.

EXAMPLE 13

TRAIL Blocking Study

[0249]Human microvascular endothelial cells of dermal origin were treated
for 16-18 hours with plasma from patients with thrombotic
thrombocytopenic purpura (TIP) or with control plasma, either alone or in
the presence of anti-TRAIL polyclonal antiserum. A 1:2000 dilution of the
antiserum was employed. The plasma was from two TTP patients, designated
#1 and #2 below, The cells employed in the assays were MVEC-1 (HMVEC
2753, purchased from Clonetics, San Diego, Calif.) and MVEC-2 (DHMVEC
30282, purchased from Cell Systems, Kirkland, Wash.). Cultures of these
cells can be maintained as described in Laurence et al. (Blood, 87:3245,
1996).

[0252]Examples of fusion proteins comprising leucine zipper (LZ) peptides
fused to the N-terminus of a soluble TRAIL polypeptide are as follows.
The leucine zipper moieties promote oligomerization of the TRAIL
polypeptides fused thereto, as described above.

[0253]An expression vector is constructed, containing DNA encoding (from
N- to C-terminus) a human growth hormone signal peptide, a leucine zipper
peptide, and a soluble human TRAIL polypeptide. The TRAIL polypeptide
comprises amino acids 95 to 281 of SEQ ID NO:2. This TRAIL polypeptide is
a fragment of the extracellular domain of human TRAIL, lacking the spacer
region, as described in example 7.

[0255]The fusion protein may comprise additional amino acid residue(s),
encoded by DNA segments that result from construction of the vector, or
that are added to facilitate vector construction. In one particular
embodiment, the tripeptide Thr-Ser-Ser is positioned between the growth
hormone signal peptide and the leucine zipper peptide. This tripeptide is
encoded by a DNA segment that comprises an Spe I restriction endonuclease
recognition site. The tripeptide Thr-Arg-Ser, encoded by a DNA segment
that comprises a Bgl II restriction site, may be positioned between the
leucine zipper and the TRAIL polypeptide.

[0256]DNA encoding the desired fusion protein is inserted into a suitable
expression vector, such as the pDC409 vector described in example 7.
CV1-EBNA cells are transformed with the recombinant expression vector,
then cultured to allow expression of the fusion protein, and
oligomerization thereof.